National Institutes of HealthNational Science FoundationFlorida State University Research FoundationState of Florida

Department of Chemistry & BiochemistryInstitute of Molecular Biophysics

National High Magnetic Field LaboratoryFlorida State University

Tallahassee, Florida

Charcterization of the M2 Proton Channel

Functional Mechanism from Influenza A

Obtained from Lipid Bilayer Preparations

Winter School January 2016

Neuraminidase

Hemaglutinin

M2

The “Spanish Flu” Pandemic of 1918-1919

Makeshift hospitals in the US

• 1/3 of the world’s population was infected

• 3% of the world’s population died (50 to 100 million people)

• In the US, 28% of the population was infected

• In the US, 500,000 to 650,000 died

• Most fatalities were healthy young adults

Brief Outline

• Intro to M2 and Influenza

• Intro to Oriented Sample solid state NMR

• M2 structure Determination in Lipid Bilayers

• Structural Comparisons

• Magic Angle Spinning solid state NMR Results

• The role of HxxxW in Proton Conductance

2

Influenza A Viral Life Cycle

M2 ProtonChannel

Adapted from Lamb & Krug, 19963

Influenza A Viral Life Cycle

Rossman & Lamb, 2011

Ç1 2 3

321

4

M S L LT

EVET

P I R N E WG CR

CNDSSDPL

VV

A AS

II G

IL

HL

IL W

IL

DR

L

N

E

T

S A

FF

KC

I

YR

FF

E

H

GL

K

RG P T GE

E

S

E

AV

S D

PV

Y R M S

E

K R

SQ QE D AD

EL

E I S HVF

M2 Proton

Channel

Amino Acid

Sequence

Amino Terminus: 2 Cys

residues form crosslinks

Amphipathic Helix that

binds lipid membrane

surface

Carboxy-

Terminus:

M1 Binding

Domain

Transmembrane Helix

essential for proton

conductance

Viral

Membrane

Aqueous Viral Interior

Aqueous Viral Exterior

0

40

80

120

160

6.5 6 5.5

H+/

tetr

amer

/s

pHex

kDa

50

252015

10

M2(22-62)

• Liposomal Assays show H+ conductance

similar to full length protein & similar

sensitivity to amantadine.

• Similar to Schnell & Chou, 2008 The

conductance domain is shown to be a tetramer

running at a slightly higher molecular weight

that the predicted 19 kDa.

SDS PAGE

6.5 6.0 5.5pH

40

80

120

160

H+ /

Tetr

ame

r/s

0

500

1000

2000

1500

H+ /

Tetr

ame

r

0-5 5 10 15 20Time (s)

M2 Conductance Domain

w/o AMT

w AMT

w/o AMT

w AMT

0

Emily Peterson and David Busath, BYUPacifichem - Honolulu - 12/16/2010

Some of the Data for the M2 Conductance Domain Proton Channel Structure

• A tetrameric structure that shows some dimer of dimer character, but only subtle evidence in the OS ssNMRspectra.

Viral Exterior

M2 Proton Channel Characterized by ssNMR in DOPC/DOPG Lipids

The structure restrained primarily by orientational restraints and refined using restrained MD all in the same lipid bilayer

The structure of the H37 tetrad and Trp41 tetrad were refined using QM/MM calculations

V27

H37

W41

Sharma et al., 2010 Science

Viral External

Pore

Viral

Internal Pore

Viral Interior

Viral Interior

Viral Exterior

His-His+ Pair

Scarce Water in pore

due to Trp41 residues

In the pore – the primary

gate for proton

conductance

Abundant Water in pore

due to Gly 34 residues

In the pore

Waters & Lipids Associated with the M2 Conductance

Domain Structure

His37 Labeled Full–Length M2 (H57,90Y)

pH 7.3 pH 6.6

Figure 5.1 Aromatic regions of 2D 13

C-13

C correlation spectra with 50ms mixing

time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)

pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold

symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch

H37) is

labeled in (C); The chemical exchange peaks are labeled in blue and the

short-distance correlations are labeled in green in (B); The additional neutral

conformation t5 is labeled in (B).

a

b

Figure 5.1 Aromatic regions of 2D 13

C-13

C correlation spectra with 50ms mixing

time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)

pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold

symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch

H37) is

labeled in (C); The chemical exchange peaks are labeled in blue and the

short-distance correlations are labeled in green in (B); The additional neutral

conformation t5 is labeled in (B).

a

b

13C (ppm)

13C

(p

pm

)1

3C

(p

pm

)

Miao et al., 2015 Structure

50 ms DARR spectra

@ -10°C In DOPC/DOPE

as in PDB: 2L0J

>>Dimer of Dimer? >>Additional Complexity

3B

KD

Sto

uff

er e

t al

., 2

00

813C-13C Correlation Spectra of His37 at lower pH

pH 6.6 pH 6.2 pH 5.8

Figure 5.1 Aromatic regions of 2D 13

C-13

C correlation spectra with 50ms mixing

time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)

pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold

symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch

H37) is

labeled in (C); The chemical exchange peaks are labeled in blue and the

short-distance correlations are labeled in green in (B); The additional neutral

conformation t5 is labeled in (B).

a

b

13C

(p

pm

)

pH 6.6 pH 6.2 pH 5.8

13C (ppm)

Miao et al., Structure, 2015

Figure 5.1 Aromatic regions of 2D 13

C-13

C correlation spectra with 50ms mixing

time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)

pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold

symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch

H37) is

labeled in (C); The chemical exchange peaks are labeled in blue and the

short-distance correlations are labeled in green in (B); The additional neutral

conformation t5 is labeled in (B).

a

b

50 ms DARR spectra

@ -10°C

Even More Complexity

3B

KD

Sto

uff

er e

t al

., 2

00

8

200220240

15N (ppm)

180 160260

His37 pH Titration of Full Length M2 in DOPC/DOPE Bilayers.

Miao et al., Structure, 2015

Charge

pH7.0 6.5 6.0 5.5 5.0 4.57.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

pKas: 6.3 ± 0.16.3 ± 0.1

5.5 ± 0.3

Broadened Lines Dynamics & Heterogeneity

>>Cooperative Protonation of His37

3B

KD

Sto

uff

er e

t al

., 2

00

815N Spin Echo Spectra of pH 6.2 spectra

Miao et al., Structure, 2015

0.167 ms

5.0 ms

2.5 ms

Heterogeneous broadening

3B

KD

Sto

uff

er e

t al

., 2

00

8Both p & t uncharged states observed at high pH

13C (ppm)

15N

(pp

m)

t1t2

t3

p1

Figure 5.1 Aromatic regions of 2D 13

C-13

C correlation spectra with 50ms mixing

time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)

pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold

symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch

H37) is

labeled in (C); The chemical exchange peaks are labeled in blue and the

short-distance correlations are labeled in green in (B); The additional neutral

conformation t5 is labeled in (B).

a a

bb

Cg/Nd1Ce1/Nd1

Cd2/Ne2Ce1/Ne2

Cd2/Ne2Ce1/Ne2

Ce1/Nd1

Cg/Nd1

Miao et al., Structure, 2015

NC zf TEDOR Spectra

1 ms mixing time

pH 7.3 & -10°C Full Length M2

3B

KD

Sto

uff

er e

t al

., 2

00

8NC Spectra of the His37 at pH 7.3 & 6.2

13C (ppm)

pH 7.3

pH 6.6

Figure 5.1 Aromatic regions of 2D 13

C-13

C correlation spectra with 50ms mixing

time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)

pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold

symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch

H37) is

labeled in (C); The chemical exchange peaks are labeled in blue and the

short-distance correlations are labeled in green in (B); The additional neutral

conformation t5 is labeled in (B).

15N (ppm)

Miao et al., Structure, 2015

195238253 170- - - -

255

170

160

180

190

200

170

160

180

260

250

160

260

250

160

170

180

15N

(p

pm

)

245

235

190

180

170

160

pH 6.2

Stable Structural Heterogeneity

Figure 5.1 Aromatic regions of 2D 13

C-13

C correlation spectra with 50ms mixing

time at 243K on His37 labeled M2FL as a function of pH. (A) pH4.5. (B) pH5.8. (C)

pH6.2. (D) pH6.6. (E) pH7.3. (F) pH8.8. The four neutral His37 with two-fold

symmetric backbone are labeled as t1, t2, t3, p in (F); The charged His37 (ch

H37) is

labeled in (C); The chemical exchange peaks are labeled in blue and the

short-distance correlations are labeled in green in (B); The additional neutral

conformation t5 is labeled in (B).

N••••H––N

M2FL NC Spectra of the His37 at pH 6.2

NP

P

NP

P

PP

• Imidazole-Imidazolium

H-bonded pairs

15N

(p

pm

)

13C (ppm)

170

160

180

190

235

245

255

170

160

180

190

200

t +

Miao et al., Structure, 2015

His Tetrad Chemistry Driven by Charge Delocalization, Structural Stability

Separated Charges:

No H-Bond

Solvated His-His+

Dong et al., Chem. Sci. RSC, 2013

His-His+ Short H-bonds Confirmed in Full Length M2

15N (ppm)

1H (ppm)

5

10

15

20

160200 180

••••

••••

Miao et al., Structure, 2015

+

+t

His+ protons exchange with water

His-His+ H-bonds associated with

high 1H and 15N frequenciesx

HN HETCOR

500 µs contact time

pH 6.2

-10°C

Multiple His-His+ States

His-His+ states exchange with

H2O-His+ states

193 ppm

188 ppm

3B

KD

Sto

uff

er e

t al

., 2

00

8Some Limits on the Exchange Rates

pH 6.2

15N (ppm)

1H (ppm)

5

10

15

20

160200 180

••

••

3600 Hz

4800 Hz

300 Hz

In 1H dimension:

4800 Hz > Exchange Rate > 0.2 Hz

Hydronium attack on the neutral His

In 15N dimension

300 Hz > Exchange Rate

3B

KD

Sto

uff

er e

t al

., 2

00

8Evidence for Short H-Bonds near Physiological Temperature

15N (ppm)

1H (ppm)

The stability of the multiple states suggests:• these states are not dictated by sidechains• not by the very stable backbone structure• but by the oligomeric helix packing

5

10

15

20

160200 180

••

••

Miao et al., Structure, 2015

At 23°C 15N frequencies associated with short H-bonds are present

At 23°C the His-His+ crosspeaks are not directly observed, but must be present since

resonces at 190 ppm are observed

Ch1 Ce1 – t1 Ce1

Ch1 Ce1 - t2 Ce1Ch1 Cg - t2 Cg

Ch1 Cd2 - t2 Ce1

Ch1 Ce1 – t1 Cd2

13C ppm

13C ppm

Evidence for both Exchange and Distances in the His37 Tetrad

Here exchange and

distances from just one

of the charged state

(Ch1) are identified.

Miao et al., 2015 Structure

pH 5.8 50 ms DARR

Sidechain Structural Dynamics at the TM – TM Interface

An Explanation for His-His+ Heterogeneity

3B

KD

Sto

uff

er e

t al

., 2

00

8HN Spectra of the His37 at pH 6.2 at -10°C (red) & 23°C (blue) – M2

15N (ppm)

1H

(ppm

)

5

10

15

20

160200 180

••

••

Miao et al., 2015 Structure

Viral Interior

Viral Exterior

His-His+ Pair

Scarce Water in pore

due to Trp41 residues

In the pore – the primary

gate for proton

conductance

Abundant Water in pore

due to Gly 34 residues

In the pore

Waters & Lipids Associated with the M2 Conductance Domain

Structure

Viral Exterior

Viral Interior

Attack by hydronium appears to

be sub-msec timescale

Conductance is known to be ~100/s

Maybe even less frequent at -10°C

A Futile Cycle in which hydronium attacks

from the external pore and the proton is

reabsorbed by a water in the aqueous pore

---

Evidence for a Futile Cycle

M2 Conductance Mechanism • Hydronium ion attack of the +2 state resulting

in a +3 state and breaking a short H-bond.

• A proton can be absorbed by

water from either the external or

internal pore

• If the proton is absorbed

into the external pore

a Futile Cycle results

• If the proton is absorbed

into internal pore a

Conductance Cycle results

Miao et al., Structure, 2015

Cholesterol Stabilizes the

Amphipathic Helix

Mixed sample of:

13C Leucine FLM2 and

13C Phenylalanine FLM2

Slices through Leu Ca

w/o AMT & w/o Cho

w AMT & w/o Cho

w AMT & w Cho

Ekanayake et al., in press, BJ

Distances in Red represent distances

From the 2L0J M2 structure

Slice through C25

Slice through C3

13CLabeled Phenylalanine FLM2

& Labeled Cholesterol

Cholesterol and M2FL

Phe Cg

Phe Cd,e

Ekanayake et al., in press, BJ

A Typical Cholesterol binding

pocket with:

• C3 close to Phe 54

• Cholesterol in Helix-Helix Crevice

• Cholesterol close to palmitylation

site, Cys50.

Fourth & Final Set of Conclusions:

• M2 has a unique Histidine Tetrad that is a His–His+ Dimer of Dimers Structure

• M2 Full Length Protein Displays Exchange and Dynamic Processes to Facilitate Proton Exchange

• M2 Conductance is a Combination of a Futile and Conductance Cycles

• Biological Chemistry Requires a Molecular Framework on which to Hang the Chemistry & Dynamics to Facilitate the Chemistry

• Solid State NMR can Uniquely Characterize the Structural and Dynamic Details in Membrane Proteins in a variety of model environments and in cell membranes

Just Some of those

who contributed so

much to my lab