Close and Allosteric Opening of the Polypeptide-Binding ... · mechanism of this allosteric...

Article

Close and Allosteric Open
ing of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP
Graphical Abstract

Highlights

d Crystal structure of an intact human BiP in the ATP-bound

state

d Crystal structure of isolated BiP-SBD with a peptide

substrate bound

d The structures provided a structural explanation for allosteric

coupling in Hsp70s

d BiP has a unique NBD-SBD interface that is highly conserved

only in eukaryotic Hsp70s

Yang et al., 2015, Structure 23, 2191–2203December 1, 2015 ª2015 Elsevier Ltd All rights reservedhttp://dx.doi.org/10.1016/j.str.2015.10.012

Authors

Jiao Yang, Melesse Nune, Yinong

Zong, Lei Zhou, Qinglian Liu

[email protected]

In Brief

Hsp70s play a key role in protein folding

and homeostasis. Yang et al. determined

structures of human Hsp70 BiP in the

ATP-bound state and the isolated SBD

with a peptide bound. These structures

and biochemical analysis revealed the

molecular mechanism of substrate

binding and allosteric coupling in

eukaryotic Hsp70s.

Accession Numbers

5E84

5E85

5E86

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http://dx.doi.org/10.1016/j.str.2015.10.012

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Structure

Article

Close and Allosteric Openingof the Polypeptide-Binding Sitein a Human Hsp70 Chaperone BiPJiao Yang,1 Melesse Nune,1,2 Yinong Zong,1 Lei Zhou,1 and Qinglian Liu1,*1Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA 23298, USA2Present address: Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA

*Correspondence: [email protected]://dx.doi.org/10.1016/j.str.2015.10.012

SUMMARY

Binding immunoglobulin protein (BiP), an essentialand ubiquitous Hsp70 chaperone in the ER, plays akey role in protein folding and quality control. BiPcontains two functional domains: a nucleotide-bind-ing domain (NBD) and a substrate-binding domain(SBD). NBD binds and hydrolyzes ATP; the sub-strates for SBD are extended polypeptides. ATPbinding allosterically accelerates polypeptide bind-ing and release. Although crucial to the chaperoneactivity, the molecular mechanisms of polypeptidebinding and allosteric coupling of BiP are poorly un-derstood. Here, we present crystal structures of anintact human BiP in the ATP-bound state, the firstintact eukaryotic Hsp70 structure, and isolated BiP-SBD with a peptide substrate bound representingthe ADP-bound state. These structures and ourbiochemical analysis demonstrate that BiP has aunique NBD-SBD interface that is highly conservedonly in eukaryotic Hsp70s found in the cytosol andER to fortify its ATP-bound state and promote theopening of its polypeptide-binding pocket.

INTRODUCTION

The key functions of the ER are folding, assembly, and quality

control for secreted and membrane proteins (Hammond and

Helenius, 1995). Binding immunoglobulin protein (BiP), an essen-

tial and ubiquitous Hsp70 molecular chaperone resident in the

lumen of ER, plays a crucial role in all of these ER functions

(Dudek et al., 2009; Hendershot, 2004).

Hsp70s are a class of conserved and abundant molecular

chaperones that play multiple essential roles in maintaining

cellular protein homeostasis by assisting protein folding, assem-

bly, translocation into organelles, and degradation (Bukau et al.,

2000; Hartl and Hayer-Hartl, 2009; Mayer and Bukau, 2005;

Young, 2010). Hsp70s have been found in the cytosol of both

prokaryotes and eukaryotes, and in all the cellular compartments

of eukaryotes including the ER and mitochondria. All Hsp70s

including BiP have two conserved functional domains: a nucleo-

tide-binding domain (NBD) at the N terminus and a substrate-

Structure 23, 2191–22

binding domain (SBD) at the C terminus (Bukau and Horwich,

1998; Mayer and Bukau, 2005). NBD binds ATP and hydrolyzes

it to ADP. SBD binds hydrophobic polypeptides in an extended

conformation as substrates (Blond-Elguindi et al., 1993; Rudiger

et al., 1997; Zhu et al., 1996). Extensive structural efforts over the

past three decades have yielded a number of isolated domain

structures from both prokaryotic and eukaryotic Hsp70s. These

structures have shown the conserved structural basis of each

domain in binding its substrates. NBD is composed of two large

lobes, between which is the nucleotide-binding site (Flaherty

et al., 1990; Mayer and Bukau, 2005). SBD is divided into two

subdomains: SBDb and SBDa (Chang et al., 2008; Leu et al.,

2014; Liebscher and Roujeinikova, 2009; Zhu et al., 1996). The

polypeptide-binding pocket is formed between two loops on

SBDb while SBDa functions as a lid covering the pocket.

The chaperone activity of Hsp70s is powered by ATP through

allosteric coupling of the two functional domains (Buchberger

et al., 1995; Mayer and Bukau, 2005). In the ADP-bound and

nucleotide-free (apo) states, the two domains have little interac-

tion (Bertelsen et al., 2009; Buchberger et al., 1995; Chang et al.,

2008; Swain et al., 2007). The polypeptide substrate-binding

properties of this state are like those of the isolated SBD, high af-

finity with both slow binding and release rates (Flynn et al., 1989;

Schmid et al., 1994). In contrast, in the ATP-bound state the

two domains are tightly coupled, which results in drastically

accelerated kinetics in both binding and release of polypeptide

substrates, although the resulting affinity is two to three orders

of magnitude lower (Schmid et al., 1994). This ATP-induced allo-

steric coupling is crucial for efficient chaperone activity (Mayer

and Bukau, 2005). Thus, the molecular mechanism of allostery

had been highly sought through obtaining crystal structures of

intact Hsp70s in the ATP-bound state. However, due to the tran-

sient nature of the ATP-bound state, only recently have the

captured DnaK-ATP structures first revealed the molecular

mechanism of this allosteric coupling in Escherichia coli (Kityk

et al., 2012; Qi et al., 2013).

However, DnaK shares only 40%–50% sequence identity to

various human Hsp70s, and more importantly, DnaK’s cellular

functions differ from those of human Hsp70s, especially BiP

(Dudek et al., 2009; Ma and Hendershot, 2004). Moreover,

previous studies have shown that the biochemical properties

of eukaryotic Hsp70s are significantly different from those of

DnaK including the kinetics for peptide substrate binding, the

molecular radius of the ATP-bound state, and ATP-induced

allosteric coupling (Mapa et al., 2010; Marcinowski et al., 2013;

03, December 1, 2015 ª2015 Elsevier Ltd All rights reserved 2191

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B

F E

C

A

D

Figure 1. Constructs for Crystallization of a

Full-Length BiP and its Isolated SBD

(A) BiP binds NR peptide through its SBD and in an

ATP-sensitive manner. Fluorescence polarization

assaywith serial dilutions of BiP proteins was used

to measure NR binding. DnaK was used as a

positive control. Error bars, SEM (n > 3).

(B) Peptide NR binding affinities for DnaK and BiP

proteins. Dissociation constants (indicated by Kd)

were calculated based on the results in (A). SEM

were calculated from at least six assays on more

than two protein purifications.

(C) Schematics of BiP domain structure and the

construct for crystallization of a full-length BiP.

The coloring of domains is: NBD (blue), linker

(purple), SBDb (green), and SBDa (red). The signal

sequence (first 24 residues) and the last 20 resi-

dues are not colored. The residue numbers

marking domains are labeled on the top.

(D) Schematics of the BiP-SBD constructs for

crystallization. Domain coloring is the same as in

(C). The Tev linker and linked NR peptide are

shown as an orange line and in cyan, respectively.

(E) Neither BiP SBD-Tev-NR nor SBD-L3,40-Tev-

NR showed appreciable binding to NR peptide.

WT BiP-SBD was used as a positive control.

Binding assay was carried out as in (A).

(F) BiP-T229Amutant binds NR peptide in an ATP-

sensitive manner like that of WT BiP, and L3,40

modification drastically compromised the NR

peptide binding. WT BiP was used as a positive

control. Binding assay was carried out as in (A).

Shi et al., 1996; Wilbanks et al., 1995), suggesting that there may

be unknown important mechanistic differences between DnaK

and eukaryotic Hsp70s. Thus, the exact molecular mechanism

of the ATP-driven allosteric coupling in human Hsp70s is ill

defined. To directly answer this question, we have solved a crys-

tal structure of an intact human BiP in the ATP-bound state, the

first eukaryotic Hsp70 structure in the ATP-bound state. More-

over, to understand the structural basis of peptide substrate

binding in the ADP-bound and apo states, we solved structures

of the isolated SBD of BiP. Together with our biochemical anal-

ysis, these structures support the hypothesis that the opening of

the polypeptide-binding pocket upon ATP binding is conserved

in BiP and, most likely, in other eukaryotic Hsp70s.

RESULTS

Human Hsp70 BiP Constructs for Crystallization StudiesTo understand the molecular mechanism of BiP peptide sub-

strate binding and allosteric regulation by ATP, we aimed to

2192 Structure 23, 2191–2203, December 1, 2015 ª2015 Elsevier Ltd All rights reserved

solve crystal structures of human BiP

in two states: the isolated SBD, which

represents the ADP-bound and apo

states, and a functionally complete BiP

in the ATP-bound state. First, we tested

whether purified human BiP binds to the

NR peptide (sequence NRLLLTG), a

well-characterized peptide substrate

for DnaK (Zhu et al., 1996). As shown in

Figure 1A, BiP binds NR peptide with good affinity: the dissoci-

ation constant (Kd) is about two times lower than that of DnaK

(Figure 1B). However, the binding kinetics, both on and off rates,

are much slower than those of DnaK (Figures S1A and S1B),

which is consistent with previous reports with other peptide sub-

strates (Marcinowski et al., 2011, 2013). Moreover, addition of

ATP drastically reduced the affinity of BiP for NR, supporting

allosteric regulation of peptide substrate binding by ATP (Fig-

ure 1A). As expected, the isolated SBD of BiP binds NR with

affinity comparable with full-length BiP (Figures 1A and 1B).

To solve a crystal structure of the isolated SBD of BiP,

we linked the NR peptide to the C terminus of BiP-SBD through

a linker containing a Tev protease digest site (sequence: SEN-

LYFQGS; Figures 1C and 1D). Linking the NR peptide to the C

terminus of BiP completely blocks the binding of free NR peptide

in solution (Figure 1E), suggesting that the linked NR is bound

to the isolated SBD as substrate. We named this construct

SBD-Tev-NR, and solved its crystal structure at 2.57 A resolution

(Table 1).

Table 1. Data Collection and Refinement Statistics

BiP-T229A L3,40 (Native) SBD-Tev-NR (Native) SBD-L3,4

0-Tev-NR (Native) SBD-L3,40-Tev-NR (Se-SAD)

Data Collection

Space group P3221 C2221 C2221 C2221

Cell dimensions

a, b, c (A) 222.468, 222.468, 209.460 36.378, 91.661, 141.797 34.536, 82.593, 150.776 34.553, 82.661, 150.840

a, b, g (�) 90, 90, 120 90, 90, 90 90, 90, 90 90, 90, 90

Wavelength 0.979 0.979 0.979 0.979

Resolution (A) 50–3.00 (3.05–3.00)a 50–2.57 (2.61–2.57)a 50–2.68 (2.73–2.68)a 50–2.69 (2.74–2.69)a

Rsym or Rmerge 0.118 (0.402)a 0.044 (0.120)a 0.041 (0.092)a 0.036 (0.097)a

I/s 25.4 (4.1)a 56.6 (16.2)a 38.6 (9.6)a 70.1 (23.0)a

Completeness (%) 97.8 (98.6)a 99.8 (98.0)a 99.8 (95.8)a 100 (100)a

Redundancy 7.3 (7.3)a 6.6 (4.2)a 4.6 (3.0)a 8.6 (6.2)a

Refinement

Resolution (A) 40.76–2.99 38.49–2.57 41.30–2.68

No. of reflections 111,753 7,908 6,391

Rwork/Rfree (%) 24.3/28.7 21.9/26.4 21.60/25.55

No. of atoms 28,436 1,891 1,803

Protein 28,147 1,838 1,787

ATP/Zn/Mg 186/24/17 – –

Water 32 48 16

B factors 76.68 30.56 56.25

Protein 76.98 30.67 56.41

ATP/Zn/Mg 49.61/79.98/55.03 – –

Water 48.51 26.34 38.46

Root-mean-square deviations

Bond lengths (A) 0.011 0.003 0.003

Bond angles (�) 1.495 0.746 0.709aValues in parentheses are for the highest-resolution shell.

To obtain a functional intact BiP in the ATP-bound state, we

took advantage of two mutations analogous to those used in

our recently reported DnaK-ATP structure (Qi et al., 2013):

T229A and loop L3,4 alternative L3,40 (Figure 1C). The BiP

T229Amutation significantly compromised the ATPase rate (Fig-

ure S1C) but maintained allosteric coupling, as shown by the

significantly reduced affinity for NR peptide in the presence of

ATP (Figure 1F). These properties of T229A are consistent with

a previously characterized BiP T229G mutant (Wei et al., 1995).

The L3,40 mutation has its L3,4 replaced with a shortened L1,2

sequence (TASDNQP/VGG). This BiP-L3,40 protein has a

drastically reduced affinity for NR peptide (Figures 1E and 1F).

Thus, this BiP-T229A L3,40 construct helped stabilize BiP in the

ATP-bound state and solved the self-association problem that

complicates crystallization. Moreover, since the first 24 residues

are a signal sequence and the last 20 residues are largely disor-

dered based on sequence alignments, both were removed to

facilitate crystallization (Figure 1C). We obtained crystals of

BiP-T229A L3,40 grown only in the presence of ATP. We solved

this BiP-ATP structure by molecular replacement, and the final

model was refined to 3.0 A resolution (Table 1).

To test the structural impact of the L3,40 modification in BiP, we

introduced it into the SBD-Tev-NR construct and solved the


crystal structure of the resulting SBD-L3,40-Tev-NR construct

at 2.68 A resolution (Table 1).

Structures of Isolated BiP-SBD with Peptide SubstrateBoundAs expected, the isolated BiP-SBD structure contains both

SBDb and SBDa subdomains (Figure 2A). The linked NR peptide

is bound to the polypeptide-binding site formed between L1,2and L3,4 on SBDb. SBDa covers the polypeptide-binding site

as a lid with the signature kink between helices aA and aB.

The Tev linker that connects the NR peptide to the C terminus

of BiP-SBD packs against helices aB and aD/E of SBDa, and

facilitates crystal contacts. Overall, the BiP-SBD structure is

highly similar to that of the isolated DnaK-SBD structure in com-

plex with NR peptide, the first isolated SBD structure (Zhu et al.,

1996), except for the Tev linker (Figures 2B and S2A). The linked

NR peptide binds to BiP in a fashion similar to that of DnaK with

almost identical main-chain conformation (Figures 2B, S2A, and

S2B). However, the register of amino acids is shifted one residue

toward the N terminus (Figures 2C and S2B): instead of Leu4 in

DnaK, Leu5 is in the center of the polypeptide-binding pocket,

which could due to the constraints from the covalent linkage of

the NR peptide to the C terminus of SBD. A similar shift is


A

E

SBDα

SBDβ

SBDα

SBDβ

N1

R2

L3

L4

L5T6

L3,4L1,2

L5,6

L4,5

L3,4L1,2

L5,6

L4,5

αA

αC

αD/E

αB

B

SBDα

SBDβ

L3,4 L1,2

L5,6

L4,5

αBαC

αD/E180

F SBDα

SBDβ

L3,4 L1,2

L5,6

L4,5

180

H V429

I463V461

S452F451

T428

I426

V432

T434

L1,2

L3,4

L7,8 L7,8

L7,8 L7,8

αA

R2

L3

L4

L5T6

N1

N1

R2

L3

L4

L5T6

DC

V429

L4

Y570

R492

G

Figure 2. Structural Analysis of Isolated BiP-SBD Structures

(A) Ribbon diagrams of the BiP SBD-Tev-NR structure. Domain coloring is the

same as in Figure 1C with the Tev linker and NR peptide shown in blue and

cyan, respectively.

(B) Comparison of the BiP SBD-Tev-NR structure with theDnaK-SBD structure

(PDB: 1DKZ). The coloring of BiP SBD-Tev-NR is the same as in (A). The DnaK-

SBD structure is shown in orange, with the bound NR peptide in purple. The

structures were superimposed based on Ca atoms of SBDb.

(C) Comparison of the bound NR peptide in the BiP SBD-Tev-NR (top) and

DnaK-SBD (bottom; PDB: 1DKZ) structures. The two structures were super-

imposed as in (B). The side chains of NR peptides are highlighted in stick

presentation, and the carbon atoms of the NR peptide are colored in gray and

orange for the BiP SBD-Tev-NR and DnaK-SBD structures, respectively.

(D) Y570 and R492 form novel hydrophobic contacts with L4 of bound NR

peptide in the BiP-SBD structure. The BiP-SBD structure is shown in ribbon

representation and colored as in (A). L4 (NR peptide), V429, R492, and Y570

are highlighted as sticks.

(E) Ribbon diagram of the BiP SBD-L3,40-Tev-NR structure. Domain coloring is

the same as in Figure 1C with the Tev linker and NR peptide in blue and cyan,

respectively.

(F) Superposition of BiP SBD-L3,40-Tev-NR (purple) with BiP SBD-Tev-NR

(domain coloring is the same as in A) based on Ca atoms of SBDb.

(G) Superposition of the NR peptides in BiP SBD-L3,40-Tev-NR (purple) and BiP

SBD-Tev-NR (cyan). The two structures were superimposed as in (F) The side

chains are shown in stick representation, with carbon atoms shown in orange

for SBD-L3,40-Tev-NR and gray for SBD-Tev-NR.

2194 Structure 23, 2191–2203, December 1, 2015 ª2015 Elsevier Ltd

observed in the isolated DnaK-SBD structure with the L3,40 modi-

fication, DnaK SBD-L3,40, in which the interdomain linker from a

symmetry mate binds to the polypeptide-binding site like a pep-

tide substrate (Qi et al., 2013) (Figures S2D and S2E). These

shifts suggest the flexibility of the polypeptide-binding site in

recognition of substrates.

In the polypeptide-binding pocket, except for V429 in BiP and

M404 in DnaK, the residues that form hydrophobic contacts with

NR peptide in BiP are identical to those in DnaK (Figure S2C)

and, more importantly, the side chains of these residues assume

almost identical conformations, suggesting high conservation

of the polypeptide-binding site among Hsp70s. Mutating V429

in hamster BiP to methionine has been shown to change the

kinetics of peptide substrate binding similar to those of DnaK

(Marcinowski et al., 2013), confirming that the difference be-

tween V429 in BiP and M404 in DnaK is the structural basis for

their different peptide-binding kinetics.

Interestingly, unlike M404 forming hydrophobic contacts with

SBDa, V429 is too small to form any contact with SBDa. Surpris-

ingly, Y570 from SBDa and R492 in L5,6 form direct hydrophobic

contacts with Leu4 of the NR peptide (Figure 2D), suggesting the

direct involvement of SBDa and L5,6 in binding peptide sub-

strates, which was not observed in any previous SBD structures.

These novel contactsmay stabilize the BiP-peptide complex and

thus explain the much slower peptide substrate-binding kinetics

of BiP than those of DnaK. The smaller side chain of V429 in BiP

may allow enough space for Y570 and R492 to contact Leu4 of

NR, which was not possible for the larger side chain of M404 in

DnaK. Mutating V429 to methionine in BiP may disrupt the con-

tacts from Y570 and R492 to NR peptide, resulting in increased

kinetics similar to DnaK. Thus, Y570 and R492 may contribute to

the special substrate-binding properties of BiP. Consistent with

this observation, the SBDa of BiP has been indicated in direct

binding of polypeptide substrates (Marcinowski et al., 2011).

Interestingly, a previous study identified two arginine residues

in SBDb of hamster BiP, R470 and R492 (R470 and R492 in hu-

man BiP), which were modified by ADP, and then destabilized

peptide substrate binding (Chambers et al., 2012). Based on

the DnaK-SBD structure, R470 (in L4,5) and R492 form contacts

with SBDa and thus stabilize the covering of the SBDa lid over

the polypeptide-binding pocket. This is also true for the BiP-

SBD structure. Consistent with this feature, R470E mutant has

a peptide-binding defect similar to the deletion of SBDa charac-

terized by increased dissociation rate. However, R492E mutant

has a surprisingly much stronger peptide-binding defect

than the deletion of SBDa, which cannot be explained by the

DnaK-SBD structure. The direct hydrophobic contacts with

bound NR peptide from R492 observed in the isolated BiP-

SBD structure may provide an explanation. Moreover, R467C

mutant in DnaK (R492 in BiP) has a similar defect in peptide sub-

strate binding as BiP R470E instead of R492E (data not shown),

supporting that the contacts between R492 and NR peptide may

be unique for BiP.

The structure of BiP-SBD with the L3,40 alternation, SBD-L3,40-

Tev-NR, is almost identical to that of BiP SBD-Tev-NR except for

(H) Comparison of NR-contacting residues (in stick representation) between

BiP SBD-Tev-NR (orange) and SBD-L3,40-Tev-NR (green). The two structures

were superimposed based on Ca atoms of SBDb.

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D

A

C

B

Figure 3. Overall Structure of BiP-ATP

(A) Ribbon diagram of human BiP-ATP structure. Domain coloring is the same as in Figure 1C. The bound ATP is in stick presentation, and its associating Zn ion is

shown as a gray ball. Left: classic front-face view of NBD; right: view orthogonal to that of the left panel.

(B) Comparison of human BiP-ATP structure with our previously published DnaK-ATP (PDB: 4JNE) structure. The domain coloring of BiP-ATP is the same as in (A)

The domain coloring for DnaK-ATP is: NBD (cyan), linker (orange), SBDb (yellow), and SBDa (gray). The superposition was based on Ca atoms. The viewpoint is

the same as in the right panel of (A).

(C) Superposition of SBDb domains from the BiP-ATP (green) and DnaK-ATP (yellow) structures. The loops are labeled. The superposition was based

on Ca atoms.

(D) A unique hydrogen bond was formed between D483 and D529 in the BiP-ATP structure. D483, D529, T527, N528, and Q530 are shown in stick presentation.

Hydrogen bonds are shown as dotted lines.

the L3,40 modification (Figures 2E, 2F, and S2F). Although the BiP

protein carrying the L3,40 alteration has very low affinity for pep-

tide substrates (Figure 1F), the linked NR peptide binds to the

polypeptide-binding site almost identically to that of SBD-Tev-

NR, with the same amino acid register and virtually identical

main-chain conformation, except that the last glycine residue

of the NR peptide is missing (Figure 2G). Except for the short-

ening of L3,4, the polypeptide-binding site of SBD-L3,40-Tev-NR

is essentially identical to that of SBD-Tev-NR (Figures 2H and

S2G). Thus, the L3,40 modification has no appreciable structural

impact on BiP-SBD.

The Human BiP-ATP StructureThere are six BiP molecules per asymmetric unit in the BiP-ATP

structure, and the six molecules are almost identical (Figures

S3A–S3D). As expected, each protomer contains NBD, linker,

SBDb and SBDa domains, and ATP (Figure 3A). Thus, this hu-

man BiP-ATP structure is the first intact eukaryotic Hsp70 struc-

ture in the ATP-bound state. Except for small changes in several

loops, the NBD and SBDb are virtually identical for the six proto-

mers (Figures S3B–S3D), whereas the SBDa regions showed

more difference although the overall differences were still small.

We mainly used protomer A for the following structural analysis

and comparison.

One interesting feature about the crystal packing of the BiP-

ATP structure is that among the six protomers, two pack as a


dimer similar to our previously published DnaK-ATP structure

(Qi et al., 2013) (Figure S3E). We have recently shown this dimer

to be essential for Hsp40 interaction (Sarbeng et al., 2015). Thus,

BiP, like DnaK, may also form a dimer in solution in the ATP-

bound state, facilitating Hsp40 interaction.

Consistent with various biochemical studies on a number of

Hsp70s including BiP (Buchberger et al., 1995; Mapa et al.,

2010; Marcinowski et al., 2011; Swain et al., 2007; Wei et al.,

1995), the domains form extensive contacts in the BiP-ATP

structure. SBDb binds on the back of NBD, contacting both

lobes, while SBDa docks on the side of lobe I with the first two

helices fused into one long helix. The highly conserved interdo-

main linker is fitted in the bottom groove between the two lobes

of NBD. Overall, the BiP-ATP structure is similar to the two

recently published DnaK-ATP structures (Kityk et al., 2012; Qi

et al., 2013) although the sequence identity is only 48%, suggest-

ing an overall conserved molecular mechanism of ATP-elicited

allosteric coupling among Hsp70 members despite substantial

evolutionary distances. For comparison we used our DnaK-

ATP structure, which is almost identical to the other available

structure. The relative orientation of the domains in BiP-ATP is

similar to that of DnaK-ATP with a rotation in SBDa (Figure 3B),

consistent with the high conservation of the NBD-SBDb interface

and relatively low conservation of the NBD-SBDa interface (see

below for detailed comparison). The NBD shares the most

resemblance with virtually identical conformation (Figure S4A),


suggesting high conservation in ATP binding. The bound ATP

molecules are superimposable, although Zn2+ replaced Mg2+

in BiP-ATP due to the high concentration of Zn2+ in crystallization

solution (Figure S4B). Moreover, BiP proteins have lower ATPase

activity in the presence of Zn2+ than that of Mg2+ (Figure S1C),

consistent with the presence of ATP in the BiP-ATP structure.

Despite these similarities, there are notable differences be-

tween the BiP-ATP and DnaK-ATP structures.

NBD

There are three insertion/deletion segments in the NBD between

BiP and DnaK: (1) b8-b9, (2) b13-b14, and (3) b15-b16 (Fig-

ure S4A), whose functions had largely been a mystery. BiP has

a longer b8-b9, but shorter b13-b14 and b15-b16. Based on

sequence alignment of these segments, Hsp70s can be divided

into two groups: eukaryotic cytosolic/ER Hsp70s (BiP-like) and

prokaryotic/mitochondrial Hsp70s (DnaK-like) (Figures S4C–

S4E). The sequences are conserved in each group. In the BiP-

ATP structure, the b8-b9 insertion is involved in the NBD-SBDa

interface, and is discussed in more detail below. b13-b14 has

been proposed to be involved in Hsp40 interaction (Ahmad

et al., 2011); thus, it may contribute to the different properties

in Hsp40 interaction. b15-b16 in DnaK contributes to the interac-

tion with the nucleotide-exchange factor GrpE (Harrison et al.,

1997), a co-chaperone that only exists in prokaryotes and eu-

karyotic mitochondria, which may explain the conservation

only in these Hsp70s.

SBD

There are apparent differences in both SBDb and SBDa,

although the overall conformations are similar to those of our

DnaK-ATP structure (Figures 3B, 3C, and S4F). Notably, there

are significant differences in the loops on the L3,4 side of the poly-

peptide-binding site (including L3,4, L5,6, and L7,8), consistent

with the high flexibility of these loops (Kityk et al., 2012), which

is further supported by the larger difference among the six pro-

moters in the BiP-ATP structure (Figures S3C–S3D). Intriguingly,

L7,8 is significantly longer in the BiP-ATP structure (Figure 3C)

while it is virtually identical in the isolated SBD structures (Fig-

ure 2B). It seems that b8 slides toward L7,8 against the rest of

the structure. This could partially be due to the insertion of the

highly conserved R532 in the La,b in BiP (see below for details).

Furthermore, D529 on b8 forms a short strong hydrogen bond

with D483 on b5 in BiP-ATP (Figure 3D), which could stabilize

the position of b8 relative to b5 in addition to the main-chain

hydrogen bonds between these two b strands. Supporting the

conformation of D529, two salt bridges are formed between

N527 and Q530. All these residues, D483, D529, N527, and

Q530, are only conserved in the eukaryotic cytosolic/ER

Hsp70s (Figure S4G), suggesting that these features of the

BiP-ATP structure may be conserved among these Hsp70s,

but not in prokaryotic/mitochondrial Hsp70s.

In SBDa, reflecting the relatively low sequence conservation,

the loops connecting the helices have different conformations

from those of DnaK-ATP (Figure S4F). Interestingly, the C termi-

nus of aA/B seems less bent than that of DnaK-ATP, which cor-

relates with the NBD-SBDa interface difference betweenBiP and

DnaK as described below.Moreover, among the six almost iden-

tical protomers in the BiP-ATP structure, SBDa has the biggest

difference (Figures S3B–S3C), further supporting the intrinsic

flexibility of SBDa.


Taken together, the differences and similarities described

above support an overall conserved but differently regulatedmo-

lecular mechanism of allostery among Hsp70s.

The Unique Role of the NBD-SBDa Interface and La,b

in Eukaryotic Hsp70 Allosteric CouplingThe distinct conformation of the BiP-ATP structure is due to the

extensive contacts between domains upon ATP binding. Like the

DnaK-ATP and Hsp110-ATP structures (Kityk et al., 2012; Liu

and Hendrickson, 2007; Qi et al., 2013), there are also three ma-

jor interdomain contacts in BiP-ATP: NBD-linker, NBD-SBDb,

and NBD-SBDa (Figure 3A). Hsp110s are distant homologs of

Hsp70s, and our previously solved Hsp110-ATP structure

suggests that Hsp110-ATP is an evolutionary vestige of

Hsp70-ATP (Liu and Hendrickson, 2007). Notably, the NBD-

SBDa interface is divergent, which is consistent with the low

sequence conservation in SBDa. Two clusters of hydrophobic

contacts are featured at this interface in BiP-ATP (Figure 4A).

The first is mediated by L533 andM541 at the N terminus of helix

aA/B. This cluster is highly conserved in DnaK-ATP (L507 and

M515) (Figure 4B), and similar contacts are observed in

Hsp110-ATP (L542 and L550) (Figure 4C). For the second clus-

ter, F548 in the middle of helix aA/B forms extensive van der

Waals contacts with six residues from NBD: F68, R74, I132,

K138, F140, and M148 (Figure 4A). This cluster is similar to

that in Hsp110-ATP, whereas this interaction is replaced by a

salt bridge between N522 and E118 in DnaK-ATP. Moreover,

all the residues in this second cluster are highly conserved in

the eukaryotic cytosolic/ER Hsp70s, but not in the prokaryotic/

mitochondrial Hsp70s (Figures 4D, S5A, and S5B). Interestingly,

three residues in NBD that form hydrophobic contacts with F548

are in the segment of b8-b9 (Figures S5B and S5C), one of the

three insertions/deletions in NBD between the eukaryotic cyto-

solic/ER and prokaryotic/mitochondrial Hsp70s (Figures S4A

and S4C). Thus, this NBD-SBDa contact may be unique to eu-

karyotic cytosolic/ER Hsp70s. Moreover, our previous muta-

tional work has shown the importance of F548 in the chaperone

activity of Ssa1, themajor cytosolic Hsp70 in yeast (Liu and Hen-

drickson, 2007), supporting the importance of this NBD-SBDa

contact.

In BiP, La,b, the small linker between SBDa and SBDb, is one

residue longer than in DnaK, with an extra arginine, Arg532 (Fig-

ure 4D). This Arg residue is highly conserved among Hsp70s

from eukaryotic cytosol and ER, but missing in prokaryotic and

mitochondrial Hsp70s (Figure 4D), which had been amystery un-

til our BiP-ATP structure. Interestingly, Arg532 forms two

hydrogen bonds with D178 from NBD in the BiP-ATP structure

(Figure 4E) while it is on the surface of the isolated SBD structure,

with no obvious function (Figure S2H), suggesting its importance

in stabilizing the ATP-bound state. Thus, we hypothesized that it

may play a role in the ATP-induced allosteric coupling of Hsp70s

from eukaryotic cytosol and ER. To test this idea, we changed it

to glutamate in BiP. This R532Emutant has normal affinity for NR

peptide in the presence of ADP (Figures 1B and 4F), consistent

with its location on the surface of isolated SBD with no apparent

role in binding peptide substrate. In contrast, in the presence of

ATP, the affinity of R532E is higher than that of wild-type (WT) BiP

(Figure 4F), suggesting that the ATP-bound state is compro-

mised by R532E. To further confirm that the ATP-induced

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E

A B C

GF

D

Time(s)1,000

Figure 4. The Unique NBD-SBDa Interface and NBD-La,b Contact in BiP-ATP

(A–C) Ribbon diagrams of NBD-SBDa interfaces in the BiP-ATP (A), DnaK-ATP (PDB: 4JNE) (B), and Sse1-ATP (PDB: 2QXL) (C) structures. NBDs are in blue

and SBDas are in red. Residues forming the two clusters of contacts are shown in stick presentation. Residues labeled in green are highly conserved between

BiP-ATP and DnaK-ATP; residues labeled in orange are conserved between BiP-ATP and Sse1-ATP.

(D) Sequence alignment among Hsp70s. Secondary structure assignments are labeled on the top with cylinder for helix and arrow for strand. R532 and F548 are

highlighted in red and green, respectively. h, human; d, Drosophila melanogaster; b, bovine; v, Virgibacillus halodenitrificans. DnaK is from Escherichia coli. Kar2,

Ssa1, and Ssc1 are from Saccharomyces cerevisiae.

(E) The unique contact of NBD-La,b in the BiP-ATP structure. R532 forms two hydrogen bonds with D178 on NBD (blue). SBDb and SBDa are in green and red,

respectively. Hydrogen bonds are shown as dotted lines.

(F) Fluorescence anisotropy assay of NR peptide-binding affinity for BiP R532Emutant.WTBiPwas used as a control. Assays were carried out as in Figure 1 in the

presence of ATP (+ATP) or ADP (+ADP).

(G) BiP R532E has a defect in releasing NR peptide upon addition of ATP. BiP proteins were incubated with F-NR peptide in the presence of ADP. After binding

reached equilibrium, ATP was added (indicated by an arrow), and the release of F-NR was monitored over time.

allosteric coupling is compromised in R532E, we assayed

bound-peptide release triggered by ATP binding. Upon ATP

addition, WT BiP released the majority of its bound NR peptide


(Figure 4G). In contrast, R532E mutant demonstrated signifi-

cantly reduced release of bound NR peptide. Thus, we conclude

that this conserved arginine is important for the ATP-induced


allosteric coupling in BiP, supporting its role in stabilizing the

ATP-bound state as observed in the BiP-ATP structure.

Consistent with the high conservation of the NBD-linker and

NBD-SBDb interfaces, these interfaces in BiP-ATP are highly

similar to those in DnaK-ATP.

Conserved Opening of the Polypeptide-Binding PocketBoth NBD and SBD of the BiP-ATP structure undergo a number

of radical conformational changes relative to the isolated domain

structures, which represent the ADP-bound and apo states.

There are several isolated NBD structures of BiP in complex

with different nucleotides including ADP and ATP (Macias

et al., 2011; Wisniewska et al., 2010). All assume virtually the

same conformation even when ATP is bound. The conformation

of these structures is basically identical to that of the isolated

bovine Hsc70 NBD structure with ADP bound, the first NBD

structure (Flaherty et al., 1990). Thus, this conformation has

been believed to be the ADP-bound conformation. In contrast,

the NBD of BiP-ATP adopts a drastically different conformation

with a rotation of more than 20� between the two lobes (Figures

5A and S6), suggesting that the SBD and its interaction with

NBD is required to stabilize the ATP-bound conformation of

NBD. Thus, ATP binding mainly induces a large rotation of the

two lobes against each other to form amore closed conformation

of the nucleotide-binding site. This is consistent with DnaK-ATP

and Hsp110-ATP (Kityk et al., 2012; Liu and Hendrickson, 2007;

Qi et al., 2013). Thismore closed conformation of NBDprovides a

suitable surface for SBD subdomains and the interdomain linker

to formextensive contacts, and thenpropagates toSBD to cause

striking conformational changes in both subdomains of SBD.

It is well established that the isolated SBD structures represent

the ADP-bound and apo states (Bertelsen et al., 2009). With NR

peptide bound, the polypeptide-binding site adopts a closed

conformation in both the BiP-SBD and DnaK-SBD structures

(Figures 2A and 2B). The peptide-binding loops, L1,2 and L3,4,

close on the bound NR peptide. Moreover, the polypeptide-

binding site in SBDb is covered up by SBDa. In contrast, the

SBDa of the BiP-ATP structure is peeled away from covering

the polypeptide-binding site (Figures 3A, 5B, and 5C). The L1,2side of the polypeptide-binding site including L1,2 and L4,5 as-

sumes a conformation nearly identical to that of BiP-SBD; in

contrast, the L3,4 side of the polypeptide-binding site is wide

open: both L3,4 and L5,6 are flipped out and away, and the L3,4side of b3 and b4 is shifted downward (Figures 5B–5E). L5,6shifted as much as 16.1 A at the Ca of R492. Thus, the van der

Waals contacts with the NR peptide from this side of the poly-

peptide-binding site were abolished (Figures 5F–5H). The open

conformation of the polypeptide-binding site is consistent with

the low affinity and fast kinetics of peptide substrate binding in

the ATP-bound state. We observed a similar open conformation

in our DnaK-ATP structure (Qi et al., 2013), suggesting an overall

conserved opening of the polypeptide-binding site elicited by

ATP binding among both prokaryotic and eukaryotic Hsp70s.

Consistent with the role of L3,4 and L5,6 in both peptide substrate

binding and ATP-induced allosteric coupling, a previous study

isolated two mutations in yeast BiP (Kar2), T473L (T453 in L3,4of BiP), and P515L (P495 in L5,6 of BiP), which showed defects

in both peptide substrate binding and allosteric coupling (Kabani

et al., 2003).


Different Hsp40 Interaction between BiP and DnaKwhen the Polypeptide-Binding Pocket Is OpenComparing the open conformation of the polypeptide-binding

pocket in BiP-ATP with the close conformation in the isolated

SBD structure, two glycine residues on L5,6, G486 and G493,

changed their backbone conformations drastically (Table S1).

We observed similar changes in DnaK-ATP with analogous

G461 and G468 (Table S1). Previously, we have shown that

these glycine residues were crucial for the ATP-induced opening

of the polypeptide-binding pocket in DnaK (Qi et al., 2013). Thus,

these two glycine residues in BiP most likely play the same

crucial role for the opening of the polypeptide-binding site

upon ATP binding. To test this hypothesis, we mutated both

G486 and G493 to proline as for the DnaK-PP mutant (DnaK-

G461P/G468P). We named this mutant BiP-PP. Like the DnaK-

PP mutant, BiP-PP has reduced affinity for NR peptide and

fast binding kinetics in the presence of ADP, as happens for

WT BiP in the presence of ATP (Figure 6A and 6B). This is

different from the WT BiP in the presence of ADP, where binding

affinity is high and binding kinetics are slow. Thus, like the

DnaK-PP mutant, the BiP-PP mutant locks BiP’s SBDb into the

ATP-bound conformation regardless of the ATP or ADP status

of the NBD. In summary, these two glycine residues have a

conserved essential role in the allosteric opening of the polypep-

tide-binding pocket in BiP as in DnaK.

It is well established that peptide substrate binding stimulates

the ATPase activity of Hsp70s (Flynn et al., 1989; Mayer and Bu-

kau, 2005), the other half of the allosteric coupling. As expected,

NR peptide stimulates the ATPase activity of BiP close to 15-fold

at 400 mM in our hands (Figure 6C). In contrast, BiP-PP showed

little appreciable stimulation byNR peptide, whereas the intrinsic

ATPase rate was similar to that of the WT protein (Figure 6C). At

the same time, we observed similar results for the DnaK-PP

mutant protein (data not shown). Therefore, peptide substrate

stimulation of the ATPase activity requires the closure of the

polypeptide-binding site as in the isolated SBD structures for

both BiP and DnaK.

The chaperone activity of Hsp70s is further regulated by two

classes of co-chaperones: Hsp40s and nucleotide-exchange

factors (NEFs) (Hartl and Hayer-Hartl, 2009; Hendrickson and

Liu, 2008; Kampinga and Craig, 2010; Mayer and Bukau, 2005;

Young, 2010). While NEFs facilitate the exchange of ADP for

ATP, Hsp40s have been shown to specifically recognize the

ATP-bound state of Hsp70s and stimulate the ATP hydrolysis

step by Hsp70s. Since these two conserved glycine residues

on L5,6 are crucial for the opening of the polypeptide-binding

pocket in the ATP-bound state, we tested whether the open

conformation of the polypeptide-binding pocket is essential for

Hsp40 interaction. ERdj3, an abundant class I Hsp40 in ER,

has been proposed to be an Hsp40 for BiP and has been shown

to stimulate the ATPase activity of BiP (Jin et al., 2009; Otero

et al., 2010; Tan et al., 2014). In our hands, ERdj3 stimulated

the ATPase activity of BiP more than 20-fold at 4 mM (Figure 6D).

Intriguingly, ERdj3 failed to show any appreciable stimulation of

the ATPase rate of BiP-PP (Figure 6D), suggesting that the open

conformation of the polypeptide-binding pocket of the ATP-

bound state is not sufficient for ERdj3 stimulation. DnaJ, an

Hsp40 co-chaperone for DnaK and founding representative of

the class I Hsp40s, stimulates the ATPase rate of DnaK robustly,

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A

ED

15.9 Å 16.1 Å

ADP

ATP

23.6

Lobe I

Lobe II

ADP

ATP

ADP (L3,4')

ATP

CB

αAαB

αAαB

αAαB

αAαB

GF H V429

I463 V461

S452F451

T428

I426

V432

T434

L3,4

L1,2

Figure 5. Comparisons of the BiP-ATP Structure with the Isolated Domain Structures

(A) Comparison of the NBD from the BiP-ATP structure with the isolated BiP NBD structure in complex with ADP (3IUC). Subdomain coloring: for NBD of BiP-ATP,

lobe I (blue), and lobe II (red); for isolated BiP NBD structure, lobe I (brown) and lobe II (green). Left: classic front-face view; right: top view of the left panel. NBDs

are in backbone worm representation and are superimposed on the basis of lobe I Ca positions.

(B) Superposition of the BiP-ATP structure to the BiP SBD-Tev-NR structure based on the Ca positions of SBDb. Domain coloring for BiP-ATP is the same as in

Figure 3A. BiP SBD-Tev-NR is colored orange with the NR peptide highlighted in cyan.

(C) Superposition of the BiP-ATP structure to the BiP SBD-L3,40-Tev-NR structure based on the Ca positions of SBDb. Domain coloring for BiP-ATP is the same as

in Figure 3A. BiP SBD-L3,40-Tev-NR is colored purple with the NR peptide highlighted in cyan.

(D and E) Close-up view of (B) and (C), respectively. Only SBDb domains are shown. The Ca atoms of R492 are shown as blue spheres.

(F–H) Comparisons of polypeptide-binding site conformations. The polypeptide-binding site for BiP-ATP (F), superposition of (F) with BiP SBD-Tev-NR structure

(G), and superposition of (F) with BiP SBD-L3,40-Tev-NR structure (H) are shown in backbone worm representations. The superposition is based on Cas in L1,2 and

L4,5. Residues that form van der Waals contacts with NR peptide in BiP SBD-Tev-NR and SBD-L3,40-Tev-NR are highlighted in stick representation.

Structure 23, 2191–2203, December 1, 2015 ª2015 Elsevier Ltd All rights reserved 2199

CA

D E

GF

B

2,000 4,000 6,000

Figure 6. The Importance of Two Conserved Glycine Residues on L5,6 of Hsp70s

(A) Peptide-binding affinity determined by fluorescence polarization assay. Assays were carried out in the presence of ADP (+ADP) or ATP (+ATP).

(B) Fluorescence anisotropy assay of peptide substrate-binding kinetics. The binding reactions of F-NR peptide were carried out in the presence of either ADP

(+ADP) or ATP (+ATP), and the measurements were started right after mixing F-NR with the indicated protein.

(C–G) NR peptide and Hsp40 stimulation of BiP and DnaK in a single-turnover ATPase assay. Fold of stimulation was calculated by setting the intrinsic ATPase

activity as 1. Error bars, SEM (n > 3). (C) NR peptide failed to stimulate the ATPase activity of the BiP-PP mutant. (D and F) Neither ERdj3 (D) nor DnaJ (F) showed

appreciable stimulation on theATPase activity of theBiP-PPmutant. (E andG) TheDnaK-PPproteinmanifested significant stimulationbybothDnaJ (E) andERdj3 (G).

close to 60-fold at 0.4 mM in our hands (Figure 6E). In contrast to

ERdj3, DnaJ stimulates the ATPase rate of DnaK-PP drastically,

about two-thirds that of WT, suggesting that the open conforma-

tion of the polypeptide-binding pocket is sufficient for DnaJ

stimulation of DnaK. Then we tested whether this different stim-

ulation is due to different Hsp40s or Hsp70s. DnaJ stimulated

BiP, although to a lesser extent than ERdj3, but failed to stimu-

late BiP-PP appreciably (Figure 6F), consistent with the ERdj3

stimulation on BiP proteins. At the same time, ERdj3 stimulated

DnaK’s ATPase activity robustly and, more interestingly, also

showed significant stimulation on DnaK-PP although to a lesser

extent than that of DnaJ (Figure 6G). Thus, this difference in

Hsp40 stimulation is most likely due to an intrinsic difference be-

tween BiP, a eukaryotic Hsp70, and DnaK, a prokaryotic Hsp70.

DISCUSSION

In this study, we present crystal structures of human BiP that

represent its two functional states: BiP-ATP (ATP-bound state)


and isolated BiP-SBD (ADP-bound state). The BiP-ATP structure

is the first intact eukaryotic Hsp70 structure in the ATP-bound

state, the allosterically active state. The overall similarity of BiP-

ATP and DnaK-ATP structures suggests an overall conserved

molecular mechanism of the ATP-induced allosteric coupling

among Hsp70s despite sequence divergence, and different

cellular locations and functions. At the same time, the isolated

SBD of BiP assumes an overall structure almost identical to that

of DnaK, suggesting an overall high conservation on peptide sub-

strate binding among Hsp70s. The unique contacts with bound

NR peptide from SBDa and L5,6 due to the smaller side chain of

V429provide amechanistic explanation for themuchslowerpep-

tide-binding kinetics of BiP than those of DnaK. Themost striking

conservation between BiP and DnaK is the open conformation of

the polypeptide-binding pocket in the ATP-bound structures and

closed conformation in the isolated SBD structures (representing

the ADP-bound state). Thus, opening of the polypeptide-binding

site in the ATP-bound state and closing in the ADP-bound state

during the chaperone cycle are conserved among Hsp70s.

All rights reserved

Although the BiP-ATP structure shares overall similarity to

the DnaK-ATP structure, there are significant differences on

the conformation of SBDb and the NBD-SBDa interface.

With more hydrophobic contacts, the NBD-SBDa interface in

BiP-ATP seems stronger than that in DnaK-ATP. Sequence

alignment suggests that this interface is more conserved among

eukaryotic cytosolic/ERHsp70s but is different fromprokaryotic/

mitochondrial Hsp70s. Moreover, R532 on La,b of BiP is highly

conserved in eukaryotic cytosolic/ER Hsp70s but is missing in

prokaryotic/mitochondrial Hsp70s. This study suggests that

R532 forms contacts with NBD and plays a role in stabilizing

the ATP-bound state in BiP. Thus, this arginine further

strengthens the NBD-SBDa interface in the ATP-bound state.

It is possible that the NBD-SBDa interface together with La,bplays a more important role in eukaryotic cytosolic/ER Hsp70s

than in prokaryotic/mitochondrial Hsp70s in the ATP-induced

allosteric coupling. The functional meaning of a stronger NBD-

SBDa interface for eukaryotic Hsp70s in the cytosol and ER

needs further exploration. It is interesting that sequence align-

ments on the regions with differences between BiP-ATP and

DnaK-ATP suggest that Hsp70s can be divided into two sub-

groups: eukaryotic cytosolic/ER and prokaryotic/mitochondrial

Hsp70s. It appears that BiP-ATP represents the eukaryotic cyto-

solic/ER Hsp70s while DnaK-ATP represents the prokaryotic/

mitochondrial Hsp70s. Interestingly, the BiP-ATP structure is

compatible with available data on its interaction with Ire1 in

unfolded protein response (Figure S4H).

Our ATPase assay suggests that closing of the polypeptide-

binding pocket is required for the stimulation of the ATPase

activity of Hsp70s by peptide substrates. Closing the polypep-

tide-binding pocket could lead to the dissociation of SBD from

NBD, and freed NBD has a faster ATPase rate (Swain et al.,

2007; Vogel et al., 2006), which could be the basis of peptide

substrate stimulation of the ATPase activity (Kityk et al., 2012;

Zhuravleva et al., 2012). In contrast, Hsp40 stimulation presents

a more complicated picture. On the one hand, neither ERdj3 nor

DnaJ showed appreciable stimulation of the ATPase activity of

BiP-PP. On the other hand, both DnaJ and ERdj3 stimulated

DnaK-PP significantly. It seems that there is significant differ-

ence between BiP and DnaK in interacting with Hsp40s. Consis-

tent with this observation, ERdj3 had been reported to show

significant interaction with BiP in the presence of ADP (Marci-

nowski et al., 2011); in contrast, several previous studies demon-

strated that DnaK has little interaction with DnaJ in the presence

of ADP (Mayer et al., 1999; Sarbeng et al., 2015; Suh et al., 1999).

It is well established that Hsp40s specifically recognize the

ATP-bound state of Hsp70s. It has been proposed that Hsp40

has two binding sites on Hsp70: one is on the NBD and the other

is on SBD, at or near the polypeptide-binding site (Davis et al.,

1999; Suh et al., 1998, 1999). For BiP, either the open conforma-

tion of the polypeptide-binding pocket is not enough for Hsp40s

to bind, or closing of the polypeptide-binding pocket is required

for the stimulation. Since ERdj3 showed significant interaction

with BiP in the ADP-bound state where the polypeptide-binding

pocket is closed (Marcinowski et al., 2011), it is possible closing

the polypeptide-binding pocket is required for ERdj3 stimulation

on BiP. This is different from the DnaK-DnaJ interaction, for

which interaction was detected only in the presence of ATP.

The ADP-bound state of DnaK with closed polypeptide-binding


pocket may not be part of the requirement for this interaction.

Thus, the open conformation of the polypeptide-binding pocket

in the ATP-bound statemay be sufficient. The functional implica-

tion of this difference in Hsp40 interaction waits for further

studies.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification

The BiP-T229A-L3,40 (residues 25–633) construct used for crystallization was

cloned into a pSMT3 vector (a generous gift from Dr. Chris Lima, Sloan-Ketter-

ing Institute). After expressing as a Smt3 fusion protein in BL21(DE3) Gold,

the fusion protein was first purified on a HisTrap column using 23 PBS buffer.

The Smt3 tag was removed by Ulp1 protease. The BiP protein was separated

from the Smt3 tag and Ulp1 protease on a second HisTrap column. After

further purification using a HiTrap Q and Superdex 200 16/60 columns, the

BiP protein was concentrated to �40 mg/ml in a buffer containing 5 mM

HEPES-KOH (pH 7.5) and 10 mM KCl, and flash-frozen in liquid nitrogen.

The two BiP-SBD constructs (residues 418–636) used for crystallization and

BiP WT and mutant proteins used in biochemical assays were cloned, ex-

pressed, andpurified in essentially the sameway as that of theBiP-T229A-L3,40.

DnaK, DnaJ, GrpE, and ERdj3 proteins were expressed and purified as

described previously (Jin et al., 2009; Kumar et al., 2011; Sarbeng et al.,

2015). Details are included in the Supplementary Experimental Procedures.

The ERdj3 expression plasmid was a generous gift from Dr. Linda Hendershot.

Crystallization, Data Collection, and Model Building

All crystals were obtained with a hanging-drop vapor diffusion method.

The BiP-T229A-L3,40 protein was diluted to 10 mg/ml with buffer A (5 mM

HEPES-KOH [pH 7.5], 10 mM KCl, 5 mM Mg(OAc)2 and 2 mM ATP), and crys-

tals were obtained at 4�C with a mother liquor containing 8%–12% polyeth-

ylene glycol 3000, 0.1 M acetate acid (pH 4.5), and 0.2 M zinc acetate. Before

being flash-frozen in liquid nitrogen, crystals were treated with 0.25% glutaral-

dehyde for at least 12 hr and cryoprotected by 15% MPD (2-methyl-2, 4-pen-

tanediol) in the mother liquor. Crystals for SBD-Tev-NR and SBD-L3,40-Tev-NR

were grown at 20�C in similar conditions: 2.0 M ammonium sulfate, 0.1 M ac-

etate acid (pH 4.5), and 2.67% acetonitrile for SBD-Tev-NR; and 2.5 M ammo-

nium sulfate, 0.1 M acetate acid (pH 5.0), and 66.7 mM sodium malonate (pH

7.0) for SBD-L3,40-Tev-NR. Both crystals were cryoprotected with 18%–20%

glycerol before being flash-frozen in liquid nitrogen. Selenomethionyl (SeMet)

protein was prepared for SBD-L3,4-Tev-NR, and crystals were obtained in the

same condition as the native protein.

All diffraction datasets were collected at Beamline X4C of NSLS, Broo-

khaven National Laboratory, at 100 K with cryostream. Indexing, integration,

and scaling of the diffraction data were performed using HKL2000. Model

building was carried out in Coot.

A 3.0-A resolution native dataset for BiP-T229A-L3,40 was collected at a

wavelength 0.979 A. Structure solution was obtained by molecular replace-

ment with Phaser using our previously solved DnaK-ATP structure (PDB:

4JNE) as search model. Refinement was carried out with Refmac. For SBD-

L3,40-Tev-NR, a single-wavelength anomalous diffraction (SAD) dataset was

collected at Se peak with a SeMet crystal. Phases were evaluated with

hkl2map, and structure was developed and refined with Phenix using a

2.68-A native dataset. A native dataset at 2.57 A resolution was collected

from an SBD-Tev-NR crystal. Structure was solved by molecular replacement

using Phaser with SBD-L3,40-Tev-NR as search model. The refinement was

carried out with Phenix.

Fluorescence Anisotropy Assays for Peptide Substrate-Binding

Affinity and Kinetics

The assay was performed as described previously (Kumar et al., 2011; Xu

et al., 2012). Details are given in Supplementary Experimental Procedures.

Single-Turnover ATPase Assay

We used single-turnover ATPase assay to determine the ATP hydrolysis rates

of BiP and DnaK. The assay was carried out as described before for DnaK (Ku-

mar et al., 2011). Details are in the Supplementary Experimental Procedures.


ACCESSION NUMBERS

Atomic coordinates and structure factors have been deposited in the RSCB

PDB under the accession numbers PDB: 5E84, 5E85, and 5E86.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

six figures, and one table and can be found with this article online at http://

dx.doi.org/10.1016/j.str.2015.10.012.

AUTHOR CONTRIBUTIONS

J.Y., L.Z., and Q.L. designed the study. J.Y. carried out most of the experi-

ments. All authors wrote or edited the manuscript.

ACKNOWLEDGMENTS

We thank Drs. Wayne Hendrickson, Elizabeth Craig, Wei Yang, Lois Greene,

Diomedes Logothetis, Tricia Serio, Young-Jai You, and Leon Avery for critically

reading themanuscript and providing insightful suggestions.We are grateful to

staff at BNLBeamline X4C for their assistance in collecting diffraction data.We

thank Dr. Linda Hendershot for the ERdj3 expression plasmid. This work was

supported byNIH (1R01GM098592 toQ.L.) and Blick Scholar Award fromVCU

(to Q.L.). L.Z. is partially supported by 1RO1GM109193 from NIH.

Received: May 29, 2015

Revised: October 2, 2015

Accepted: October 12, 2015

Published: November 19, 2015

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Structure, Volume 23

Supplemental Information

Close and Allosteric Opening

of the Polypeptide-Binding Site

in a Human Hsp70 Chaperone BiP

Jiao Yang, Melesse Nune, Yinong Zong, Lei Zhou, and Qinglian Liu

Figure S1

Figure S1, related to Figure 1. The peptide NR binding kinetics and intrinsic ATPase activity.(A), Comparison of NR peptide binding kinetics between BiP and DnaK. Right after model peptide F-NR was mixed with either BiP or DnaK (at 30 µM), fluorescence polarization readings were recorded overtime to track binding kinetics.(B), Comparison of the kinetics of NR peptide release between BiP and DnaK. 30 µM BiP or DnaK wasincubated with 20 nM F-NR for more than 5 hours to allow binding to reach equilibrium. Then, unlabeledNR was added to a final concentration of 10 µM to compete off the bound F-NR. Florescence polarizationreading was measured over time to record the release of F-NR. On the right are calculated release rates(koff).(C), The intrinsic ATPase activity of the wild-type (WT) BiP and BiP-T229A proteins in the presenceMg2+ or Zn2+. Single-turnover ATPase assay was performed with BiP proteins at 20°C as described in theMaterials and Methods in the presence of either Mg2+ or Zn2+. Percentage of ATP hydrolysis was calculatedfor each time point, and plotted against time. The rate of ATP hydrolysis (kcat) was deduced by fitting first-order rate equation using nonlinear regression (GraphPad Prism).

0 5000 1000015000200002500020

40

60

80

100

120

Time (second)

Po

lari

zati

on

(m

P) BiP

DnaK

0 2000 4000 6000 80000

50

100

150

Time (second)

Po

lari

zati

on

(m

P) BiP

DnaK

A. B.

Koff (x10-3/sec)

DnaK 1.22±0.0097

BiP 0.149±0.00068

Kcat (10-3/min)

WT BiP (Mg2+) 12.38±0.233

BiP-T229A (Mg2+) 2.61±0.025

WT BiP (Zn2+) 6.48±0.161

BiP-T229A (Zn2+) 0.224±0.014

C.

0 300 600 900 12000

25

50

75

100

WT (Mg2+)T229A (Mg2+)WT (Zn2+)

Time (min.)

% A

TP

hyd

roly

sis

0 5000 10000 15000 200000

25

50

75

100

WT (Mg2+)T229A (Mg2+)WT (Zn2+)T229A (Zn2+)

Time (min.)

% A

TP

hyd

roly

sis

A.

SBDα

SBDβ

SBDα

SBDβ

G. V429

I463V461

S452F451

T428

I426

V432

T434

V429

I463V461

S452F451

T428

I426

V432

T434

L1,2 L1,2L3,4

L3,4

Figure S2

H.

R532

R532

V389

NC

CNL390

L391

L392N1 R2

L3

L4

L5

T6

S388

C.

M404

F426

S427

A429

V436T403V407

T409I401

L3,4L5,6

L1,2

B.

F. D. E.

D393

Figure S2, related to Figure 2. Structural analysis of isolated BiP-SBD structures(A), Comparison of the BiP SBD-Tev–NR structure with the isolated DnaK SBD structure (PDB: 1DKZ).Orthogonal view of Figure 2B. The SBDβ is almost identical to that of DnaK (rmsd = 0.477 Å); whereas SBDαis rotated slightly starting from the C-terminal half of helix αB. Thus, the rmsd of the SBDα is larger (2.316 Å).(B), Superposition of the bound NR peptide in BiP SBD-Tev–NR and DnaK SBD structures. NR are shownin the same way as Figure 2C. The SBDβs of BiP (green) and DnaK (orange) are shown in ribbon diagram.(C), Superposition of NR peptide interacting residues from isolated BiP SBD-Tev–NR structure withthose from the isolated DnaK SBD structure. Domain coloring and structure superposition were the same asin Figure 2B. The side-chains of the residues that contact the NR peptide are in stick representation.(D), The inter-domain Linker from a symmetry mate binds to the polypeptide-binding site in the DnaKSBD-Lˊ3,4 structure like a peptide substrate. The segment of the inter-domain Linker analogous to the NRpeptide is shown: D393, L392, L391, L390, V389, and S388 are analogous to N1, R2, L3, L4, L5, T6, and G7of the NR peptide, respectively.(E), Superposition of (D) and the NR peptide from the BiP SBD structure (the top panel of Fig. 2C). Thetwo structures were superimposed based on Cα atoms of SBDβ. Interestingly, the register of amino acids forthese two peptides are similar. At the same time, the N-C orientation of the peptide segment in D is reversedcompared to those in the WT DnaK SBD and BiP SBD structures, further supporting the flexibility of thepolypeptide-binding pocket.(F), Superposition of BiP SBD-L3,4’-Tev-NR structure with the BiP SBD-Tev–NR structure. Orthogonalview of Figure 2E. rmsd for SBDβ = 0.723 Å; rmsd for SBDα = 1.275 Å.(G), Comparison of NR-contacting residues (in stick representation) between BiP SBD-Tev–NR (orange)and SBD-L3,4’-Tev-NR (green) structure. Left, BiP SBD-Tev–NR; right, SBD-L3,4’-Tev-NR. The sameresidues in both structures form hydrophobic contacts with the NR peptide, and these residues have almostidentical conformation(H), Arg532 is on the surface of the BiP-SBD structure. The coloring BiP-SBD is the same as Figure 2A.R532 is highlighted in stick presentation. The right panel is a close-up view of the left panel.

Figure S3

A. B.

C.

D. E.

Figure S3, related to Figure 3. Protomers in the BiP-ATP crystal.(A), There are six protomers in the asymmetric unit of the BiP-ATP crystal. Each protomer is colored witha different color: green, purple, red, blue, yellow and orange, respectively.(B), Superposition of the six protomers in the asymmetric unit of the BiP-ATP crystal. Protomer coloring isthe same as in (A).(C), Orthogonal view of (B) from the right side.(D), Closed-up view of the SBDβ after the six protomers were superimposed as in (B). The coloring of theprotomers is the same as in (A).(E), The top two protomers in (A) form a dimer similar to that of the DnaK-ATP crystals. The twoprotomers in (A) are colored in green and purple, and the two protomers in DnaK-ATP are colored in red andblue, respectively. The green protomer of BiP-ATP was superimposed with the red protomer of DnaK-ATP.

Figure S4

A. B.

13

14

1516

89

D.

hBiPdBiPKar2hHsp70bHsc70dHsp70 Ssa1MtHsp75 Ssc1DnaKvHsp70

KPYIQVDIGGGQTKTFAPEEI 145KPHISVDTSQG-AKVFAPEEI 145KPAVEVSVKGE-KKVFTPEEI 165KPKVQVSYKGE-TKAFYPEEI 119RPKVQVEYKGE-TKSFYPEEV 119KPRIRVEYKGE-RKSFYPEEV 119KPQIQVEFKGE-TKNFTPEQI 117DAWVEAH-----GKLYSPSQI 166 DAWVEAR-----GQTYSPAQI 143DAWVEVK-----GQKMAPPQI 115DAWVEVK-----GSKLAPPQV 115

8 9

Mito.

pro

cytosol

ER

C.

13 14

hBiP FDVSLLTID--NG--VFEVVATNGD 250dBiP FDVSLLTID--NG--VFEVVATNGD 250Kar2 FDVSLLSIE--NG--VFEVQATSGD 270hHsp70 FDVSILTID--DG--IFEVKATAGD 225bHsc70 FDVSILTIE--DG--IFEVKSTAGD 225dHsp70 FDVSVLTIE--DG--IFEVKATAGD 225Ssa1 FDVSLLSIE--DG--IFEVKATAGD 222MtHsp75 FDISILEIQ--KG--VFEVKSTNGD 270Ssc1 FDISILDID--NG--VFEVKSTNGD 247DnaK FDISIIEIDEVDGEKTFEVLATNGD 224vHsp70 FDISIIEVADVDGETQFEVLATNGD 224

F.

αA/B

αCαD/E

E.

QARIEIESFY----EGEDFSETLT 323QVRIEIESFF----EGDDFSETLT 323STRIEIDSFV----DGIDLSETLT 343QASLEIDSLF----EGIDFYTSIT 298QASIEIDSLY----EGIDFYTSIT 298QASIEIDSLF----EGVDFYTSVT 298QTSVEIDSLF----EGIDFYTSIT 295QTDINLPYLTMDSSGPKHLNMKLT 347STEINLPFITADASGPKHINMKFS 324QTDVNLPYITADATGPKHMNIKVT 301QTEVNLPYITADNTGPKHLNVKVT 301

15 16

cytosol

Mito.

pro

ER

G. 5

hBiP HLLGTFDLTGI 487dBiP HLLGKFDLTGI 487Kar2 NLLGKFELTGI 507hHsp70 NLLGRFELSGI 464bHsc70 NLLGKFELTGI 464dHsp70 NSLGKFELSAI 464Ssa1 NLLGKFELSGI 461MtHsp75 KLLGQFTLIGI 508Ssc1 KLIGNFTLAGI 485DnaK KSLGQFNLDGI 462vHsp70 KSLGRFDLADI 462

8

GNKNKITITNDQNRLTPE 536GNKEKIVITNDQNRLTPE 536GKSESITITNDKGRLTQE 556GKANKITITNDKGRLSKE 513GKENKITITNDKGRLSKE 513GKENRITITNDKGRLSKE 513GKSNKITITNDKGRLSKE 510GREQQIVIQSSGG-LSKD 557NKDSSITVAGSSG-LSEN 534GKEQKITIKASSG-LNED 511GKEQSIVIKASGG-LSDE 511

cytosol

Mito.

pro

ER

K123E121

N104

Q109P106

H.

Figure S4, related to Figure 3. Detailed comparison between the BiP-ATP and DnaK-ATP (pdb code:4JNE) structures.(A), Superposition of the NBDs from the BiP-ATP (blue) and DnaK-ATP (cyan) structures. The βstrands involved in insertions/deletions are labeled.(B), Superposition of the bound ATP molecules from the BiP-ATP and DnaK-ATP (purple) structures.The two ATP molecules are superimposable; Zn2+ ion (grey) in BiP-ATP structure replaced Mg2+ ion (green)in the DnaK-ATP structure.(C-E), Sequence alignments of the regions in NBD involved in insertions/deletions. The β strands forsecondary structure are specified by arrows on top of the sequences for BiP-ATP, and under the sequencesfor DnaK-ATP if there is difference from those of BiP-ATP. The insertions/deletions are highlighted in cyan.h, human; d, Drosophila melanogaster; b, bovine; v, Gram-positive bacteria Virgibacillus halodenitrificans.DnaK is from E.coli. Kar2, Ssa1 and Ssc1 are from yeast saccharomyces cerevisiae.(F), Comparison of SBDα domains from the BiP-ATP (red) and DnaK-ATP (grey) structures.(G), Sequence alignment of the β5 and β8 segments from SBDβ. The strands are labeled as arrows on topof the sequences. D483 in β5, T527, N528, D529, and Q530 in β8 of BiP are highlighted in purple.(H), The location of BiP residues involved in Ire1 interaction. The BiP-ATP structure is shown in ribbondiagram with the same domain coloring as Figure 3a. The residues involved in interacting with Ire1 arehighlighted in stick representation. It has been shown that the essential role of BiP in unfolded proteinresponse of ER is through suppressing the activation of Ire1 by directly binding to Ire1 under normalcondition(Bertolotti et al., 2000; Liu et al., 2000; Ma et al., 2002; Okamura et al., 2000). A study in yeastshowed that the molecular form of BiP that interacts with Ire1 is the ATP-bound conformation withoutpeptide substrate, the same conformation as our BiP-ATP structure. Moreover, this study suggested that Ire1binds to the lobe IB of BiP’s NBD including residues N83, R85, Q88, N100, and D102 (N104, P106, Q109,E121 and K123 in human BiP)(Todd-Corlett et al., 2007). These residues are clustered on the surface of NBDin the BiP-ATP structure. Thus, it is possible for Ire1 to bind to these residues without interfering the ATP-bound structure of BiP. Interestingly, these residues are in close proximity to the NBD-SBDβ and NBD-SBDα interfaces with D102 (K123 in BiP) on the edge of the NBD-SBDα interface. E121 and K123 are closeto the β8-β9 segment, which has the special insertion for eukaryotic cytosol/ER Hsp70s and is involved in theunique NBD-SBDα contacts observed in the BiP-ATP structure. Thus, it is conceivable that the BiP-Ire1interaction is regulated by allosteric coupling of BiP, which is supported by another recent studydemonstrating that peptide substrate binding allosterically dissociates the BiP-Ire1 interaction(Carrara et al.,2015).

Figure S4

Figure S5


NRITPSYVAFTPEGERLIGD 78NRITPSYVAFTADGERLIGD 78NRITPSYVAFTDD-ERLIGD 98NRTTPSYVAFTDT-ERLIGD 55NRTTPSYVAFTDT-ERLIGD 55NRTTPSYVAFTES-ERLIGD 55NRTTPSFVAFTDT-ERLIGD 52ARTTPSVVAFTADGERLVGM 99SRTTPSVVAFTKEGERLVGI 76DRTTPSIIAYTQDGETLVGQ 53ARTTPSIIAYTDDGETLVGQ 53

68 74

F548

K138

I132

F140 8

9

Mito.

pro

cytosol

ER

A.

C.

Figure S5, related to Figure 4. The hydrophobic contacts formed by F548 in BiP-ATP structure areconserved in Hsp70s from eukaryotic cytosol and ER.(A-B), Sequence alignments of the segments in NBD containing residues forming hydrophobiccontacts with F548 in BiP-ATP. The residues that form hydrophobic contacts with F548 in the BiP-ATPstructure are highlighted in red. h, human; d, Drosophila melanogaster; b, bovine; v, Gram-positive bacteriaVirgibacillus halodenitrificans. DnaK is from E.coli. Kar2, Ssa1 and Ssc1 are from yeast saccharomycescerevisiae.(C), F548 forms hydrophobic contacts with three residues on β8-β9 of NBD, I132, K138 and F140.F548, I132, K138, and F140 are shown in stick presentation.


KPYIQVDIGGGQTKTFAPEEISAMVLTKMKE 155KPHISVDTSQG-AKVFAPEEISAMVLGKMKE 155KPAVEVSVKGE-KKVFTPEEISGMILGKMKQ 175KPKVQVSYKGE-TKAFYPEEISSMVLTKMKE 129RPKVQVEYKGE-TKSFYPEEVSSMVLTKMKE 129KPRIRVEYKGE-RKSFYPEEVSSMVLTKMRE 129KPQIQVEFKGE-TKNFTPEQISSMVLGKMKE 127DAWVEAH-----GKLYSPSQIGAFVLMKMKE 176 DAWVEAR-----GQTYSPAQIGGFVLNKMKE 153DAWVEVK-----GQKMAPPQISAEVLKKMKK 125DAWVEVK-----GSKLAPPQVSAEVLKKMKK 125

132 138 140 148

8 9

Mito.

pro

cytosol

ER

B.

ADP

ATP

23.6°

Lobe II

Lobe I

Figure S6

Figure S6, related to Figure 5. Structural comparison of NBDs. Same as Figure 5A except that the

superposition was based on the Cα positions of Lobe II. Within each lobe, there is little difference

between the BiP-ATP and the isolated NBD structures (rmsd for lobe I and II are 0.844 and 0.595 Å,

respectively).

Backbone conformation, φ (°), ψ (°)

Gly486 in BiP(G461 in DnaK)

Gly493 in BiP(G468 in DnaK)

BiP-ATP -73.8, -24.6 -84.2, 163.2

DnaK-ATP -66.4, 168.6 -81.8, 126.6

BiP-SBD 77.9, 37.2 104.1, 0.4

DnaK-SBD 83.5, 13.1 79.8, 3.9

BiP-SBD-L3,4 96.8, 12.9 89.4, -12.4

DnaK-SBD-L3,4 65.6, 51.5 61.4, 29.2

Table S1, related to Figure 5 and 6

Comparison of phi (φ) and psi (ψ) angles

Supplemental Experimental Procedures

Protein expression and purificationAll of the DnaK proteins used in biochemical assays were expressed and purified as described

previously (Kumar et al., 2011). Briefly, all the mutants were cloned into a dnak expression plasmid pBB46 witha C-terminal hexahistidine tag, and expressed in a dnak deletion strain BB205 (camRkanR) (Burkholder et al.,1996) to avoid contamination of wild-type DnaK protein from regular E.coli expression strains. Both pBB46 andBB205 are generous gift from Dr. William Burkholder. DnaK proteins were purified on a HisTrap columnfollowed by a HiTrap Q. The final proteins were concentrated to > 10 mg/ml and flash frozen in liquid nitrogenbefore storing in -80 freezer.

DnaJ and GrpE were purified as described previously (Kumar et al., 2011; Sarbeng et al., 2015).Briefly, Smt3-DnaJ fusion protein was first purified on a HisTrap column with buffer containing Hepes-KOH,pH 7.2, 300 mM KCl, and 10% glycerol. After Smt3 tag was removed by Ulp1, it was further purified on asecond HisTrap column, and a Superdex 200 16/60 column. GrpE was purified the similar way with 2XPBSbuffer. Both proteins were concentrated to > 10 mg/ml, flash frozen in liquid nitrogen, and stored at -80freezer.

The ERdj3 expression plasmid was a generous gift from Dr. Linda Hendershot. ERdj3 was expressedand purified as described previously (Jin et al., 2009) with modifications. Briefly, 2 M urea was included in thelysis buffer during purification on a HisTrap column. Before flash frozen in liquid nitrogen, purified ERdj3 wasdialyzed against buffer containing 25 mM Hepes-KOH, pH7.5, 200 mM NaCl, 20% glycerol and 0.02% TritonX-100.

Fluorescence anisotropy assays for peptide substrate binding affinity and kineticsThe assay was performed as described previously (Kumar et al., 2011; Xu et al., 2012). Briefly, F-NR

peptide, NR peptide (sequence: NRLLLTG) labeled with a fluorescein at the N-terminus, was purchased fromNEOBioscience. To determine binding affinity in the presence of ADP, serially diluted BiP or DnaK proteinswith buffer B (25 mM Hepes-KOH, pH 7.5, 100 mM KCl, 10 mM Mg(OAc)2, 10% glycerol, and 2 mM DTT)containing 100 µM ADP were incubated with F-NR peptide (final concentration 20 nM) for 3-5 hours (at least 5hours for BiP proteins) to allow binding to reach equilibrium. Fluorescence anisotropy measurements collectedon a Beacon 2000 instrument (Invitrogen) were fitted to a one-site binding equation using PRISM (GraphPad) todeduce dissociation constants (Kd). For the binding in the presence of ATP, BiP proteins were first diluted withbuffer B. ATP was added to a final concentration of 2 mM, and incubated for 2 min to allow ATP binding. F-NRpeptide was added to a final concentration of 20 nM, and fluorescence anisotropy measurements were carriedout after 15 minutes incubation since it usually takes 10-15 min for the binding to reach equilibrium but beforeany significant ATP hydrolysis occurs.

For the measurements of peptide binding kinetics, BiP or DnaK proteins were first diluted to theindicated concentrations 20μM or 30 μM with buffer B either with ADP or ATP, and incubated for 2 min toallow nucleotide binding. Then, F-NR peptide was quickly added to start binding, and fluorescence anisotropymeasurements were read every 10s (DnaK proteins) or 30 s (BiP proteins) to track binding.

Single-turnover ATPase assayThe assay was carried out as described before for DnaK (Kumar et al., 2011; Sarbeng et al., 2015).

Briefly, 20 µg of either BiP or DnaK was diluted with buffer C (25 mM Hepes-KOH, pH 7.5, 100 mM KCl, 10mM Mg(OAc)2, and 2 mM DTT), and then incubated with 25 µCi of [α-32P] ATP (NEG503H250UC, 3000Ci/mmol; Perkin Elmer) with addition of 20 µM unlabeled ATP on ice for 1 min to allow ATP binding toHsp70s. The BiP-ATP or DnaK-ATP complex was quickly isolated from free ATP on a spin column pre-equilibrated with Buffer B, aliquoted and frozen in liquid nitrogen. Each reaction was started by mixing equalvolumes of the ATP complex with NR peptide, DnaJ or ERdj3 in indicated concentrations, and incubated at20°C or 15°C (for DnaJ stimulation). After stopping the reactions at indicated time points with stop buffer (1M formic acid, 0.5 M LiCl and 0.25 mM ATP), PEI-cellulose thin-layer chromatography plates (Sigma-Aldrich)were used to separate ATP from ADP. The amount of radioactive ATP and ADP were visualized and quantifiedwith a Typhoon phosphorimaging system (GE healthcare). The rate of ATP hydrolysis (kcat) was deduced usinga first-order rate equation by nonlinear regression (GraphPad Prism). For assays with ERdj3, buffer C withoutDTT was used, and DTT was removed from DnaK and BiP proteins before forming complexes with ATP.

Supplemental References

Bertolotti, A., Zhang, Y., Hendershot, L.M., Harding, H.P., and Ron, D. (2000). Dynamic interaction of BiP and

ER stress transducers in the unfolded-protein response. Nature cell biology 2, 326-332.

Burkholder, W.F., Zhao, X., Zhu, X., Hendrickson, W.A., Gragerov, A., and Gottesman, M.E. (1996). Mutations

in the C-terminal fragment of DnaK affecting peptide binding. Proceedings of the National Academy of

Sciences of the United States of America 93, 10632-10637.

Liu, C.Y., Schroder, M., and Kaufman, R.J. (2000). Ligand-independent dimerization activates the stress

response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum. The Journal of biological

chemistry 275, 24881-24885.

Ma, K., Vattem, K.M., and Wek, R.C. (2002). Dimerization and release of molecular chaperone inhibition

facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. The

Journal of biological chemistry 277, 18728-18735.

Okamura, K., Kimata, Y., Higashio, H., Tsuru, A., and Kohno, K. (2000). Dissociation of Kar2p/BiP from an

ER sensory molecule, Ire1p, triggers the unfolded protein response in yeast. Biochemical and biophysical

research communications 279, 445-450.

Carrara, M., Prischi, F., Nowak, P.R., Kopp, M.C., and Ali, M.M. (2015). Noncanonical binding of BiP ATPase

domain to Ire1 and Perk is dissociated by unfolded protein CH1 to initiate ER stress signaling. eLife 4.

Todd-Corlett, A., Jones, E., Seghers, C., and Gething, M.J. (2007). Lobe IB of the ATPase domain of Kar2p/BiP

interacts with Ire1p to negatively regulate the unfolded protein response in Saccharomyces cerevisiae. Journal of

molecular biology 367, 770-787.

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