TheStructureoftheMammalianSignalRecognitionParticle (SRP ... · by the SRP cycle (3, 4). SRP...

The Structure of the Mammalian Signal Recognition Particle(SRP) Receptor as Prototype for the Interaction of SmallGTPases with Longin Domains*

Received for publication, November 18, 2005, and in revised form, January 25, 2006 Published, JBC Papers in Press, January 26, 2006, DOI 10.1074/jbc.M512415200

Oliver Schlenker, Astrid Hendricks, Irmgard Sinning, and Klemens Wild1

From the Biochemie-Zentrum der Universitat Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany

The eukaryotic signal recognition particle (SRP) and its receptor(SR) play a central role in co-translational targeting of secretory andmembrane proteins to the endoplasmic reticulum. The SR is a het-erodimeric complex assembled by the two GTPases SR� and SR�,which is membrane-anchored. Here we present the 2.45-A struc-ture of mammalian SR� in its Mg2�GTP-bound state in complexwith the minimal binding domain of SR� termed SRX. SR� is amember of the Ras-GTPase superfamily closely related to Arf andSar1, while SRX belongs to the SNARE-like superfamily with a foldalso known as longin domain. SRX binds to the P loop and theswitch regions of SR�-GTP. The binding mode and structural sim-ilarity with other GTPase-effector complexes suggests a co-GAP(GTPase-activatingprotein) function for SRX.Comparisonwith thehomologous yeast structure and other longin domains reveals aconserved adjustable hydrophobic surface within SRX which is ofcentral importance for the SR�-GTP:SRX interface. A helix swap inSRX results in the formation of a dimer in the crystal structure.Based on structural conservation we present the SR�-GTP:SRXstructure as a prototype for conserved interactions in a variety ofGTPase regulated targeting events occurring at endomembranes.

Nascent chains of secretory and membrane proteins are targeted viathe ribonucleoprotein particle SRP2 and the interaction with its recep-tor (SR) to the translocation machinery within the endoplasmic reticu-lum (ER) membrane (1, 2). The function of the SRP system is describedby the SRP cycle (3, 4). SRP recognizes N-terminal hydrophobic signalsequences as soon as they emerge from the ribosomal polypeptide exittunnel. The complex is targeted to the SR at the membrane, and theribosome-nascent chain (RNC) complex is transferred to the translo-con. Upon GTP hydrolysis in SRP and SR the complex dissociates.The eukaryotic SR consists of the two GTPases SR� and SR� (1, 5).

SR� is a multidomain SRP GTPase with a characteristic low affinity fornucleotide (�10 �M) and is stable in the nucleotide-free form (6–8).The C-terminal part of SR� (FtsY in archaea and eubacteria) contains

the stable NG domain, which binds to the respective NGdomain of SRP(SRP54, Ffh in eubacteria). Several structures of the isolated NGdomains revealed the basis for the SRP GTPase cycle (for reviews, seeRefs. 2–4 and 9), and the complex of the two NG domains shows aremarkably symmetric heterodimer with the nucleotides in direct con-tact at the center of the interface (10, 11). The N-terminal part of SR� isresponsible for tethering SR� to the ERmembrane bound SR� (12). Theinteraction localizes to the globular SRX domain of SR� comprising thefirst 130 residues (13). SRX has been described as effector for SR� andonly binds to the GTP-bound form of the GTPase (13, 14). The SRXdomain belongs to the SNARE-like superfamily including the N-termi-nal domains of non-syntaxin SNAREs, also known as longin domains(13, 15). Longin domains have been proposed to regulate a variety ofmembrane trafficking processes (16). Members of this superfamily withknown three-dimensional structures include the SNAREs Sec22b (17)and Ykt6 (18), the component SEDL of the transport protein particle(TRAPP) (19), and the clathrin adaptor proteins AP-� and AP-N�

(20, 21).SR� is a classical small Ras-GTPase most similar to Arf (ADP-ribo-

sylation factor) and Sar1 (secretion-associated and Ras-related 1) pro-teins with an accordingly low KD of �30 nM for GTP (6, 22). Phyloge-netically, SR� together with Arf and Sar1 separated from other smallRas-GTPases already in the earliest branching event indicating the func-tional importance of an ancestral SR� in eukaryotic evolution (23). Typ-ical for small GTPases, SR� is characterized by five conserved sequenceelements (G1–G5), which form the nucleotide-binding site (24–26).The so-called switch I (residues 85B to 96B in SR�; to distinguishbetween SR� and SRX we use the subscript letters B and X, respec-tively), interswitch (97B to 115B) and switch II (116B to 130B) regions areknown to change most in conformation during a GTPase cycle (26). Inthe switch II region of SR�, the position of a critical catalytic residue(Gln-61 in Ras, Gln-71 in Arf) is occupied by a histidine (His-119B),which is conserved among the SR� and in the Sar1 family (His-79). Aspecial feature of SR� is its predicted membrane spanning helix, whichis unique but dispensable for SR function (27). In comparison, proteinsof the Arf and Sar1 family have an extra N-terminal helix that becomeseither myristoylated in Arf (28) or is preceded by an N-terminal hydro-phobic patch in Sar1 (29). TheGTPases are anchored in theGTP-boundstate to their target membrane.SR� does not hydrolyze GTP (30, 31) and also not in complex with

SR� or SRX alone (Refs. 13 and 22 and this study). Like for other smallRas-GTPases, a GTPase-activating protein (GAP) and a guanine nucle-otide exchange factor (GEF), which stimulate the low intrinsic GTPaseactivity and the release of GDP, respectively, are necessary to drive theGTPase cycle (26, 32). RNCs interact with SR� in its GTP-bound state(22). GTP binding to SR� is stimulated by the translocon and is requiredto induce signal sequence release from SRP (33). The GAP function fortrypsin-digested SR heterodimers that retain SR� and the N-terminal

* This work was supported by the Deutsche Forschungsgemeinschaft (SFB352 andSFB638) and by European Union Network Grant QLK-3CT-2000-00082 to (I. S.). Thecosts of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2FH5) have been deposited in the ProteinData Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, NewBrunswick, NJ (http://www.rcsb.org/).

1 To whom correspondence should be addressed. Tel.: 49-6221-544785; Fax: 49-6221-544790; E-mail: [email protected].

2 The abbreviations used are: SRP, signal recognition particle; SR, SRP receptor; GAP,GTPase-activating protein; ER, endoplasmic reticulum; RNC, ribosome-nascent chain;TRAPP, transport protein particle; Arf, ADP-ribosylation factor; Sar1, secretion-associ-ated and Ras-related 1; GEF, guanine nucleotide exchange factor; r.m.s.d., root meansquare deviation; SNARE, soluble NSF attachment protein receptor(s) (where NSFindicates N-ethylmaleimide-sensitive factor).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 13, pp. 8898 –8906, March 31, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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fragment of SR� including SRXhas been attributed to theRNCcomplex(22). Interestingly, a GAP function of the RNC for the isolated SR�

could not be found (30, 31). In the yeast system, theGEF activity for SR�

has been assigned to the two orthologues (Sbh1p, Sbh2p) of the Sec61�

subunit of the translocon (34) and point mutations in the cytoplasmicloops of the yeast translocon severely affect the co-translational trans-location pathway (35). However, the molecular details for the initiationof GTP hydrolysis and the release of GDP in SR� remain so far unclear.Here we describe the crystal structure of the mammalian SR�-GTP:

SRX receptor complex. The comparison of the structure with the yeasthomolog provides detailed insights into the family of the eukaryotic SR.The homology to other small GTPases and the underlying principles ofregulation together with previous biochemical data allows attributing aco-GAP function to SRX for SR�-GTP.We analyze the fundamentals ofthe longin domain family and suggest the interaction of small GTPasesand longin domains to be important for targeting of large complexes orvesicles to the endomembrane system.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The His6-tagged N-terminal176 amino acids from human SR� (including SRX, residues 1–130)together with mouse SR� lacking the N-terminal transmembraneregion (here referred to as SR�, residues 58–269) were expressed as abi-cistronic construct from vector pET16b (Stratagene) in BL21(DE3)Escherichia coli cells (Stratagene). The protein was purified via affinitytag purification using Ni2�-loaded chelating Sepharose Fast Flow beads(Amersham Biosciences). The protein was further purified via ionexchange chromatography (Q- and SP-Sepharose) and finally via sizeexclusion chromatography (Superdex 200, Amersham Biosciences)using a low salt buffer (10 mM Tris/HCl pH 8.0, 150 mM NaCl, 5 mM

MgCl2, and 1 mM dithiothreitol). The same buffer was also used forcrystallization trials.

Crystallization and Data Collection—Initial crystallization condi-tions were obtained from hanging drop vapor diffusion at 20 °C usingthe WIZARD sparse matrix screen (Emerald BioStructures) and a pro-tein concentration of 12 mg/ml by mixing the protein in a 1:1 ratio with

reservoir. Crystals grew within 4–6 weeks over a reservoir containing100mM sodium citrate, pH 5.5, 2.0 M (NH4)2SO4, and in the presence of100 mM guanidinium chloride. The leaf-shaped crystal plates belong tospace group I222 and diffract to 2.45 Å. The asymmetric unit containsone SR�-GTP:SRX heterodimer corresponding to a solvent content of55% and aMatthews coefficient of 2.7 Å3/Da. Crystals were flash-frozenin liquid nitrogen using 20% (v/v) glycerol as cryo-protectant. Diffrac-tion data were measured at the European Synchroton Radiation Facilityin Grenoble, France at beamline ID14–4. Data were collected at 100 K

FIGURE 1. Overall structure and SRX topology. A, the structure of the mammalian SR�-GTP:SRX monomer is shown as a ribbon diagram with SR� in cyan, SRX in green, and Mg2�GTPas a ball-and-stick model. The termini and secondary structure elements are labeled. Disordered parts of the complex are indicated by dashed lines. B, SRX topology. Secondarystructure elements are shown in green (�-strands) and magenta (�-helices). The termini and secondary structures are labeled.

TABLE 1

Data collectionWavelength (Å) 0.979Resolution range (Å) 50–2.45Completeness (%)a 99.4 (100.0)Rsym (%)a 5.7 (42.4)�I/��a 25.0 (3.3)Space group I222Unit cell dimensions (Å) a � 68.1

b � 118.3c � 120.4

Refinement statisticsResolution range (Å) 20–2.45Total reflections 18,124Working set 17,202Test set 922

Number of refined atomsProtein 2500Mg2�GTP 35Water molecules 104

B-factor (Å2) 56.5R-factor (%)b 19.3Rfree (%)b 23.2r.m.s.d. bond length (Å) 0.01r.m.s.d. bond angle (°) 1.55Ramachandran plot (%)Most favored region 90.9Additionally allowed region 8.8Generously allowed region 0.4Disallowed region 0.0

a Values in parentheses are for the highest resolution shell: 2.49–2.45 Å.b R� ��Fobs� � �Fcalc��/�h�Fobs�. R-factor and Rfree were calculated from the workingand test reflection sets, respectively. Rfree was calculated with 5% of the data.

The Structure of the Mammalian SRP Receptor

MARCH 31, 2006 • VOLUME 281 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8899

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and a wavelength of 0.979 Å and processed using the HKL programpackage (36). Data statistics are listed in Table 1.

Structure Determination and Analysis—The structure was deter-mined by molecular replacement using the �-subunit of the yeast SR�-GTP:SRX complex (Protein Data Bank code: 1NRJ) as a searchmodel inAMoRe (37).Model buildingwas donewith programO (38). Themodelincluding 104watermoleculeswas refined at 2.45Å resolution using theCNS package (39) to an R-factor of 19.3% and an R-free factor of 23.2%(Table 1). Residues 41X-47X, 131X-176X, 208B-219B, and 248B-254B arenot ordered and therefore missing in the model. All structural figureswere created with program PyMOL. Program DALI (40) was used tosearch the Protein Data Bank for proteins similar to SR� and the anglebetween the two �1X helices within the SR�-GTP:SRX “dimer” wasdetermined with program DYNDOM (41). Fig. 5 was done with theprogram ALSCRIPT (42).

RESULTS AND DISCUSSION

Overall Structure—The overall structure of the refined SR�-GTP:SRX model is depicted in Fig. 1A. SR� is a typical small GTPase andfeatures a classical Rossmann foldwith a central six-stranded (�1B–�6B)mixed �-sheet packed in between five helices. SR� reveals highest sim-ilarity to the GTP-bound structures of Sar1 in complex with Sec23/Sec24 (43) (r.m.s.d.: 1.30 Å over 143 C�-positions) and Arf1 (44)(r.m.s.d. of 1.50 Å over 150 C�-positions) reflecting their evolutionaryneighborhood (23). Besides the N-terminal membrane anchoringregions, the most striking structural difference between SR� and Arf orSar1 is an insertion between helix �4B and strand �6B (37 residuescompared with Sar1). Helix �4B is extended by two turns and protrudes

from the protein core as described earlier (13). The insertion is partiallydisordered and no particular function has been attributed to it so far. Inthe SR�-GTP:SRX complex SR� is in a state not competent for GTPhydrolysis as the catalytic histidine residue (His-119B) is pointing awayfrom the active site (see below).The SRX domain (Fig. 1, A and B) belongs to the mixed �/� class

proteins sharing topology (��) and fold of the SNARE-likeprotein superfamily including the N-terminal domains of non-syntaxinSNAREs (longin domains). The fold is defined by a three-layer architec-ture with a central five-stranded antiparallel �-sheet packed againsthelix �1X on the concave side of the �-sheet and two C-terminal anti-

FIGURE 2. The SR�-GTP:SRX dimer. The dimer formed by the SRX domains of twoSR�-GTP:SRX complexes as observed in the crystal structure reveals a domain swap ofhelix �1X (rotation is indicated) and the formation of a trans �-sheet between themerged �3x-�4x strands. The 2-fold axis between two monomers is highlighted in cyan.

FIGURE 3. The SR�-GTP:SRX interface. The interface is divided into three layers (top, center, and bottom). A, prominent features of the interface are: salt bridge between Arg-34X andAsp-72B (top), hydrogen bonds between helix �1x and switch I (top center), Ile-94B in a hydrophobic groove of SRX that is schematically denoted by black lines and the conservedGly-12X (green sphere) at the tip of the �1X-�2X hairpin in the center of the interface (center), hydrophobic interactions (black lines) and hydrogen bond between Asn-101x and Ser-98B

(bottom). Hydrogen bonds involving main chain atoms are not shown. B, detailed stereo views of the SR�-GTP:SRX interface. Residues discussed under “Results and Discussion” arehighlighted in sticks (either side chains or complete residues). Polar interactions are represented by dashed lines. The three layers are separated in the figure.



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parallel helices �2X and �3X on the other side (secondary structurenumbering is according to the Structural Classification of Proteins(SCOP) nomenclature, which is different to the nomenclature used forthe yeast structure). At the N terminus the two anti-parallel �-strands�1X-�2X are connected by a conserved �-hairpin. Helix �1X locatesalmost perpendicular to the�-strands on the concave side and connectsthe peripheral �-strands of the �-sheet (�2X and �3X). The helix flank-ing loop regions are not conserved and only partially visible in the struc-ture. Strands�3X,�4X, and�5X are connected by short�-hairpin struc-tures. The central strand �5X is followed by the long helix �2X, the�2X-�3X loop in the plane of the �-sheet, and the C-terminal helix �3Xrunning anti-parallel to helix�2X.Helix�2X is kinked andwraps aroundthe convex side of the �-sheet like a clamp and helix �LX is inserted inthe �2X-�3X loop.The mammalian SR�-GTP:SRX complex forms a crystallographic

dimer due to an interaction of the SRX domains involving a domainswap of helix �1X (�50° rotation around helical N terminus) and theformation of a continuous trans �-sheet (Fig. 2). Here, strands �3X-�4Xof one “monomer” merge and align anti-parallel across the dimer inter-face. Dimerization leads to an additional buried interface of �1000 Å2

between the two SR�-GTP:SRX monomers. To analyze the oligomer-ization state of mammalian SR�-GTP:SRX in solution, we performed asedimentation equilibrium experiment by analytical ultracentrifugationand determined a KD of 270 �M for the dimer (data not shown). Wecannot directly conclude from this result in solution to the state of thecomplete SR complex at the membrane, since full-length SR�� isanchored to the membrane in vivo. The SR�-GTP:SR� complex (with-out the transmembrane region) showed a tendency for aggregation andthe KD could not be determined. Therefore, the physiological relevancefor the dimerization of SR�-GTP:SRX is not clear. The dimer might beas well enforced by crystal packing. The crystal symmetry favors thedomain swap of the flexibly linked helix �1 (see below) due to sterichindrance. The simultaneous formation of the trans �-sheet stabilizesoligomerization by main chain hydrogen bonding. Interestingly, thecomparisonwith the yeast structure (monomer, see below) showed thatthe domain-swapped helix �1X of the second SRX molecule of themammalian receptor superimposes with its corresponding position inthe yeast monomer. Therefore, a “monomeric” mammalian receptorcomplex is used for further analysis.

The SR�-GTP:SRX Interface—The SR�-GTP:SRX interface involvesthe predominant effector-binding region of Ras-like GTPases (45) (Figs.1 and 3). The buried surface between SR�-GTP and SRX is 1850 Å2,which is similar to the yeast structure and other GTPase-effector com-plexes (13). SR� contributes to the interfacewith its G1 element (P loop,GLCDSGKT), switch I, interswitch, and switch II regions. The completeswitch I region snugly binds into a hydrophobic groove of SRX andspans the whole interface. This groove is situated between theamphipathic helix�1X and the hydrophobic concave surface of the SRX�-sheet. Although the protein interface forms a continuous surface,three regions of SRX organized in three layers contribute to the inter-face (Fig. 3): (i) helix �1X, (ii) the �-hairpin between strands �1X and�2X, and (iii) the �2X-�3X loop including the short helix �LX.

In the top layer, the amphipathic helix �1X binds the switch I and IIregions and the P loop of SR�. The side chain of the conserved Asn-30Xforms hydrogen bonds to the side chain Gln-91B (not conserved) andthemain chain of Thr-92B in switch I. One helical turn further, Arg-34Xforms a salt bridge to Asp-72B in the P loop bridging the active site andforming a hydrogen bond to the side chain of Thr-92B. Three residues ofhelix �1X (Ile-33X, Leu-37X, and Leu-38X) are part of a hydrophobicpocket, which accommodates Ile-94B and the aliphatic part of Gln-91B

in the center of the interface. Leu-38X forms an additional hydrophobicinteraction with Leu-122B of switch II.In the central layer, SRX exclusively interacts with the switch I region

of SR�. The central hydrophobic pocket is completed by Val-14X andthe aliphatic part of Lys-10X. The conserved�-hairpin between�1X and�2X contributes a number of hydrophilic interactions, which are sur-rounded by a hydrophobic rim. All hydrophilic interactions are estab-lished bymain chain atoms of the�-hairpin, which contains a conservedglycine (Gly-12X) at the tip. The carbonyl oxygen of Lys-10X forms ahydrogen bond to the amide nitrogen of Ile-94B. The carbonyl oxygen ofGly-11X approaches theMg2� binding site in SR� and forms a hydrogenbond with the side chain of Ser-93B, which is essential for Mg2� coor-dination. Residues Gly-12X to Val-14X form a short stretch of an anti-parallel trans �-sheet with residues Gln-91B to Asp-89B of switch I.

In the third layer, SRX binds to the switch I and interswitch regions ofSR�. Interactions are formed by the �2X-�3X loop including the shorthelix �LX. Three hydrophobic side chains (Ala-103X, Leu-104X, andLeu-107X) from helix �LX interact with residues Phe-79B, Val-80B, Leu-83B, and the hydrophobicmethyl group of Thr-84B from switch I as wellas with Ala-99B and Ile-100B from the interswitch region. Hydrophilicinteractions are established by main chain atoms of Ala-103X and Leu-

FIGURE 4. Comparison of mammalian and yeast SR�-GTP:SRX complexes. A, super-position of the mammalian and yeast SR�-GTP:SRX complexes based on SR�. The yeaststructure (Protein Data Bank code: 1NRJ) is shown in gray, yeast insertions in black, andmammalian insertions in blue. Insertions mentioned under “Results and Discussion” arelabeled. B, close up of the superposition showing the active site of SR�. The interactionbetween the P loop and helix �1X is indicated. The catalytic histidine is pointing awayfrom the active site and hydrogen bonded to the switch II region in both structures.



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107X, which hydrogen bond to the main chain of Ser-98B and the gua-nidinium group of Arg-88B, respectively. The layer is completed by theinteraction of the side chains of Asn-101X and Ser-98B.

Comparison with SR�-GTP:SRX from Yeast—The structures ofmammalian and yeast SR�-GTP (13) are conserved (r.m.s.d. of 1.16 Åover 158 C�-positions, yeast is distinguished in the following by a “y”subscript). Differences include the lengths of the �-strands �2B and �3Bthat are almost twice as long inmammals and helix�4B that is two turnsshorter (Fig. 4A).In contrast, there are significant differences in SRX (r.m.s.d. of 1.81 Å

over 75 C�-positions) (Fig. 4A). In the yeast structure there is no helixswap leading to a SR�-GTP:SRX dimer. Instead, the central �-sheet ofSRXy is extended by one strand (�3Xy) between helix �1Xy and strand�4Xy (�4Xy corresponds to strand �3X in our structure), which appar-ently stabilizes the position of helix �1X and thereby prevents dimerformation. While helix �1X and the �1X-�2X hairpin in the interfacesuperimpose very well, the central �-sheet and the connected helices�2X and �3X do not. Differences increase with distance from the SR�-GTP:SRX interface.Yeast SRX shows two major insertions (Fig. 4A). A 20-residue inser-

tion elongates the central �-sheet by introducing the sixth �-strand(�3Xy: Glu-46Xy to Ala-49Xy) and a loop touching helix �2Xy on theconvex side of the �-sheet. A 15-residue insertion changes the confor-mation of the loop between helices �2X and �3X and the herein insertedhelix �LX is not present. The C-terminal helices (�4Xy and �3X) do notalign, which might be due to a truncation of this helix in the yeaststructure. With its insertions, yeast SRX appears unusual comparedwith other SRX domains.

The observed structural differences between mammalian and yeastSRX are reflected by the low degree of conservation on the sequencelevel (14.2% identity, Fig. 5). Low sequence conservation is a generalfeature of the SRX family (13). One functionally important exception isthe conserved Gly-12X in the �1X-�2X hairpin (Figs. 3 and 5). It facili-tates the �-hairpin turn and a bulky side chain would sterically interferewith binding of SR�. Position and amphipathic character of the impor-tant helix�1X are conserved. Asn-30X is conserved between human andyeast and interactswith SR� by hydrogen bonding to the switch I region.A polar residue one turn further appears to occupy a crucial positionwithin helix�1X. Arg-34X forms a salt bridge with Asp-72B in the P loopand thereby influences the position of the catalytic histidine (His-119B)with respect to the active site of SR� (Fig. 4b). Although this salt bridgeis not conserved, a polar interaction is observed in the yeast structurebetween Ser-35Xy and Gln-47By within the P loop, suggesting a similarrole.

SRX as Effector for SR�—SRX occupies large parts of a typical GAPbinding site (45) as it interacts with the P loop and the switch regions ofSR�-GTP resulting in the stabilization of switch II. However, in theSR�-GTP:SRX complex the catalytic histidine (His-119B) in switch II ofSR� (Gln-61 in Ras, Gln-71 in Arf) is in a “resting” position pointingaway from the active site (Fig. 4b), the characteristic arginine finger of aGAP (46) is not present, and the complex is stable when bound to GTP.Therefore, the SR�-GTP:SRX complex is not a GTPase:GAP complex,and for the stimulation of GTP hydrolysis an additional binding partneris needed. The RNC has been shown to stimulate GTP hydrolysis ofSR�-GTP:SRX (22). However, the RNC does not act as a GAP for SR�-GTP alone (30). Therefore, the SRX domain can be assigned as co-GAP

FIGURE 5. Alignment of longin domain sequences. The alignment is based on structures (bold sequences) or secondary structure predictions (nonbold type). Known structuresinclude human and yeast SRX, mouse Sec22b (Protein Data Bank code: 1IFQ, chain A), yeast Ykt6 (1H8M), mouse SEDL (1H3Q), human AP2-�2 (1GW5, chain S), and AP2-N�2 (1GW5,chain M). The structures of �-COP (hCOPZ), �-COP (hCOPD), and the SNARE hVamp7 are not known. Sequence numbering and secondary structure assignment are shown for humanSRX above the aligned sequences (�-strands in green, �-helices in orange). The secondary structure is indicated in all sequences. The conserved glycine in the �1X-�2X loop is markedin red. The residue causing a conserved anomaly in strand �2X is indicated in green, and the critical polar position in �1X hydrogen bonding to the P loop in SR� is highlighted in blue.



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for SR� which fulfils one part of the GAP function by stabilizing switchII. Examples for a split GAP function have been reported before. TheGAP for the �-subunit of a heterotrimeric G protein (Gi�1) also stabi-lizes the switch regions, but the arginine finger is supplied in cis by anadditional domain of the GTPase (47). A unique feature of the Arf1:ArfGAP1 structure is the exclusive stabilization of the switch II region(48). The switch I region is recognized by the heptameric coat proteincomplex (COPI) (49), which is found to stimulate GTP hydrolysis (48).Most likely an arginine finger is needed to trigger GTP hydrolysis inArf1 (48), which might be the case as well in SR�.

The co-GAP function can be envisaged by a comparison of SR�-GTP:SRX with the structure of the Ras-GDP-AlF3:RasGAP transition-statecomplex (50).When SR� is superimposedwith Ras, the loop of RasGAPcontaining the arginine finger (Arg-789RasGAP) fits between SR� andSRX (Fig. 6A). The only sterical clash concerns the arginine finger itself,which would interfere with the salt bridge between Arg-34X and Asp-72B (Fig. 6, A and B). In addition, the Ras-GDP-AlF3:RasGAP complexcontains a second arginine (Arg-903RasGAP) in close proximity to Arg-34X (Fig. 6B). Arg-903RasGAP forms a salt bridge to Glu-63Ras in theswitch II region of Ras thereby stabilizing the switch II region. In SR�-

FIGURE 6. Split GAP model for SR� activation. The model is based on the superposition of the respective GTPases within SR�-GTP:SRX and the Ras-GDP-AlF3:RasGAP complex (gray,Protein Data Bank code: 1WQ1). A, SR�-GTP:SRX is shown together with the finger loop of RasGAP containing the arginine finger Arg-789RasGAP. The loop fits between SR� and SRX,and the arginine finger interrupts the Arg-34X-Asp-72B salt bridge. B, superposition of the active sites. Arg-903RasGAP occupies a similar position as Arg-34X. In the activatedRas:RasGAP complex, the arginine binds to Glu-63Ras (position Ser-121X in SRX), and the catalytic Gln-61Ras is bound to the nucleophilic water in the active site. C, model ofGAP-activated SR�-GTP:SRX. Arg-34X could bind to Ser-121B in a similar way as Arg-903RasGAP to Glu-63Ras. His-119B is rotated into the active site. The arginine finger could beprovided by the RNC-SRP complex.



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GTP:SRX the catalytic residue His-119B is hydrogen bonded to the cor-responding residue of Glu-63Ras (Ser-121B) (Fig. 6B).The comparison of SR�-GTP:SRX with the Ras-GDP-AlF3:RasGAP

complex suggests that upon the insertion of an arginine finger into theGTP binding pocket the salt bridge between Arg-34X and Asp-72B canbe disrupted. The liberated Arg-34X may then swing from the P looptoward Ser-121B in switch II forming a hydrogen bond (Fig. 6C). His-119B would therefore be released, the catalytic water can be positioned,and hydrolysis occurs. Mutants in which the salt bridge is disrupted(Asp-72B3Gly and an Arg-34X3 Ala) still form the SR�-GTP:SRXcomplex (data not shown) indicating that the missing GAP is essentialto stimulate GTP hydrolysis. The large conformational changes that aretypically observed in the switch regions upon GTP hydrolysis areexpected to disrupt the SR�:SRX interface and lead to the dissociationof the SR complex (13). In the context of the SRP cycle this could happeneither before or after signal peptide release from SRP.

Longin Domains Revisited—SRX belongs to the superfamily ofSNARE-like proteins with the longin domain fold (15). Sequencehomology within the superfamily is low (Fig. 5), but the structuralhomology is high (Fig. 7) as illustrated by the comparison of SRX withSEDL (19), with the SNAREs Sec22b (17) and Ykt6 (18), and the �2(N-terminal domain) and �2 adaptins (20). To determine conservedelements within the longin domain fold we prepared a structure basedsequence alignment of structurally known longin domains and ofimportant longin domain candidates (�-COPI, N�-COPI, VAMP7; Fig.5). Among longin domains with known structures, SRXy reveals specificinsertions like strand �3Xy, whereas the mammalian structure is closerto other members of the superfamily.Longin domains share the �� topology as described for SRX

(Fig. 1B). The glycine residue (Gly-12X in SRX) in the �1-�2 hairpin ishighly conserved (Fig. 5), and the hairpin adopts a similar conformationin all longin domain structures. Only in SEDL this glycine is exchanged

FIGURE 7. Comparison of longin domains andflexibility of helix �1. Longin domain structuresare depicted in ribbon and surface representations(Protein Data Bank codes are given). SRX is ori-ented to show the surface interacting with SR�. Allstructures are oriented accordingly. The structuresof longin domains other than SRX have beendetermined as monomers or in context of the AP2trunc (N�2 and �2). The color code corresponds tohydropathicity (60). The different orientations ofhelix �1 are indicated by a gray line. The hydropho-bic grooves in SRX and SRXy are marked by blackarrows. Hydrophobic patches are shown by blueand white arrows, respectively. Previouslydescribed hydrophobic patches are boxed (SEDL(19), Sec22b (17), Ykt6 (18)). The r.m.s.d. values ofall longin domains in respect to SRX are given.



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for an aspartate, and the change is compensated by adjustments in theadjacent�-strands. Ykt6 comprises a unique insertion of three residues.Helix �1 is an essential component of the longin domains (see below).The amphipathicity of helix �1 is highly conserved, while there is noconservation on the sequence level and the length ranges from three(SRX) to six turns (Ykt6). The orientation of helix �1 with respect to thecentral �-sheet varies in the different longin domains (Fig. 7). Flexibilityis reflected by elevated temperature factors in the loops connecting helix�1 to the �-sheet (not shown) and in the SRX structure the flexibility isresponsible for dimer formation by the swap of helix �1X (Fig. 2).A conserved�-sheet anomaly (down-up-up-down) is the insertion of

a bulky hydrophobic residue (Leu-15X in SRX, Fig. 5) within strand �2.It seems to be important for stabilizing the protein core and indicates anevolutionary relationship between the longin domains. The C-terminalhelix �3 differs in length and orientation between the individual struc-tures and superimposes best for Sec22b, SEDL, and SRX. Helix �3 istruncated in the longin domains of the AP2 complex (N�2, �2), whichaccording to secondary structure predictions is also the case in other APcomplexes and the COPI complex (N�-, �-COPI) (Fig. 5). Here, thelongin domain fold is extended by a �-hairpin structure followed byanother helix forming a fourth layer in the back of the longin domainfold (not shown). The length and the conformation of the loop regionsvary significantly (Fig. 7).

GTPase:Longin Domain Complexes at Endomembranes—The local-ization of longin domains at the endomembrane system correlates withthe presence of small membrane-associated GTPases like the Arf andSar1 proteins, which are the closest relatives of SR�. The structuralconservation and the co-localization strongly suggest that other GTP-ase:longin domain interactions may exist. Two hydrophobic patchesflanking helix �1 were noticed previously in longin domain structuresand were proposed as protein-protein interaction surfaces (17–19).Interestingly, these patches are conserved in structurally determinedlongin domains (Fig. 7). In the SR�-GTP:SRX complex, SR� binds tothis interaction surface. SR� intercalates its switch I region betweenhelix �1X and the SRX �-sheet; one of the helix flanking hydrophobicpatches is extended and forms a hydrophobic groove (Figs. 3 and 8). Infree longin domain structures the hydrophobic groove is absent (Fig. 8).The opening of the groove can be envisaged by rolling the conservedamphipathic helix�1 onto the second hydrophobic surface patch on theother side of the helix. The flexibility of helix �1 is therefore a prereq-uisite for the interaction of longin domains with their respectiveGTPase.While the conservation of the hydrophobic patches suggests a similar

mode of GTPase:longin domain interaction, the low degree of conser-vation reflects the special adaptations of the individual systems. For

example, in all known longin domains the equivalent position of Arg-34X within helix �1X (Figs. 3 and 4B) seems to be occupied by a chargedor polar residue (Fig. 5). In the respective GTPases the same is true forthe residue at the position equivalent or adjacent to Asp-72B in the Ploop. Therefore, a polar contact between helix �1 and the P loop mightbe present in all GTPase:longin domain interactions. As discussed forthe co-GAP function of SRX (see above), the residues corresponding toArg-34X could also participate in the stabilization of the switch IIregions of the respective GTPases. The mammalian SR�-GTP:SRXcomplex can thus be regarded as a structural prototype for a GTPase:longin domain interaction. Although there is no direct experimentalproof, to our knowledge this idea does not contradict any previous data.Structures of longin domains other than SRX have been determined

as monomers (Sec22b, Ykt6, SEDL) or in context of the AP adaptin“trunc” complex. All clathrin adaptor complexes (AP1, -2, -3, -4) andCOPI share a tetrameric trunc organization that consists of two large, amedium, and a small subunit (51). COPI andAP complexes contain twocopies of longin domains (N�- and �-COPI, and AP-N� and -�, respec-tively). In the structure of theAP2 complex (20), the two longin domainsform the core of the trunc with the respective �1 helices being in closeproximity. Therefore, a tandem GTPase:longin domain interactionmight be an important feature in all these complexes.

AMolecular Explanation for a Genetic Disease—The GTPase:longindomain concept offers a structural explanation for the occurrence ofspondyloepiphyseal dysplasia tarda, an X-linked skeletal disorder char-acterized by a short trunk (52). Point mutations in the human SEDLprotein seem to be involved in a defect in cartilage transport from the ER

FIGURE 8. An adjustable hydrophobic surfacewithin longin domains. SR�-bound SRX and freeSEDL are shown in the same orientation with semi-transparent surfaces. Helix �1 and the �1-�2 hair-pin are labeled. Colors are according to hydro-pathicity and hydrophobic groove and patchesare indicated. A, SRX surface as prototype for aGTPase-bound longin domain. The switch I regionof SR� (blue ribbon) binds into the hydrophobicgroove created by the “packing” defect of helix�1X and the central SRX �-sheet. Ile-94B is insertedinto a pocket in the center of the interface. B, SEDLas an example for a free longin domain. The hydro-phobic groove is not present.

FIGURE 9. Superposition of SRX and SEDL. SRX is shown in green and SEDL in magenta.Secondary structure assignment is given for human SRX. The pathogenic point mutation(Asp-47SEDL3 Tyr) within helix �1 at the protein surface is indicated.



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to the Golgi apparatus (53). The yeast homologue of SEDL (Trs20p) hasbeen shown to be part of the highly conserved transport protein particleI (TRAPP I) that is required to tether ER-derived vesicles to the Golgi(54) and consists of ten subunits (55). When the structure of SEDL issuperimposed with SRX in the SR�-GTP:SRX complex (Fig. 9), thepathogenic Asp-473 Tyr mutation in human SEDL would be locatedon the protein surface within helix �1X in close proximity to the cata-lytic residue His-119B and the interacting Ser-121B of SR� (see Fig. 4B).There is no structure of the corresponding SEDL:GTPase complex;however, Ypt1p has been shown as the TRAPP interactingGTPase (55–57) and according to ourmodel Gln-67 andArg-69 in Ypt1p could forma favorable interaction with Asp-47SEDL. Thus, themutationmost likelydisturbs theGTPase regulation by interferingwith the positioning of thecatalytic residue.

CONCLUSIONS

The structure of the mammalian SR�-GTP:SRX complex reveals theoverall features of a GTPase-effector complex. Although the sequenceidentity is very low ((13) this study), the interaction is conservedbetween mammals and yeast. The mode of interaction, previous bio-chemical data (22, 30), and the structural comparison with the Ras:RasGAP complex (50) point to a co-GAP function of SRX for SR�. SRXstabilizes the switch II region but leaves room for an additional GAP,most likely the RNC-SRP complex, to insert an arginine-finger andstimulate GTP hydrolysis, which in turn would lead to the dissociationof the SR�:SRX complex.Small membrane-bound GTPases play a central role in all transport

and targeting events at endomembranes (58, 59). Given the importanceof ribosome targeting for all protein secretion systems and the role ofSR� in this process (23), the SR�-GTP:SRX complexmay be regarded asa structural and functional prototype for GTPase:longin domain inter-actions. The regulation of complex formation is indicative of a controlpoint in the assembly of large transport complexes at the respectiveendomembrane system.The relevance of this concept needs to be testedexperimentally in the future. The molecular explanation given for thegenetic disease spondyloepiphyseal dysplasia tarda might be the firsttest case.

Acknowledgments—We thank all staff from beamlines ID14 and ID29 forexcellent support at the European Synchrotron Radiation Facility inGrenoble,France.We gratefully acknowledge JacekMazurkiewicz andDr. Karsten Rippe(Kirchhoff-Institut fur Physik, Universitat Heidelberg) for the analytical ultra-centrifugation experiments.

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Oliver Schlenker, Astrid Hendricks, Irmgard Sinning and Klemens WildPrototype for the Interaction of Small GTPases with Longin Domains

The Structure of the Mammalian Signal Recognition Particle (SRP) Receptor as

doi: 10.1074/jbc.M512415200 originally published online January 26, 20062006, 281:8898-8906.J. Biol. Chem.

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