Change in allosteric network affects binding affinities of PDZ domains: analysis through perturbation response scanning

The allosteric mechanism plays a key role in cellular functions of several PDZ domain proteins (PDZs) and is directly linked to pharmaceutical applications; however, it is a challenge to elaborate the nature and extent of these allosteric interactions. One solution to this problem is to explore the dynamics of PDZs, which may provide insights about how intramolecular communication occurs within a single domain. Here, we develop an advancement of perturbation response scanning (PRS) that couples elastic network models with linear response theory (LRT) to predict key residues in allosteric transitions of the two most studied PDZs (PSD-95 PDZ3 domain and hPTP1E PDZ2 domain). With PRS, we first identify the residues that give the highest mean square fluctuation response upon perturbing the binding sites. Strikingly, we observe that the residues with the highest mean square fluctuation response agree with experimentally determined residues involved in allosteric transitions. Second, we construct the allosteric pathways by linking the residues giving the same directional response upon perturbation of the binding sites. The predicted intramolecular communication pathways reveal that PSD-95 and hPTP1E have different pathways through the dynamic coupling of different residue pairs. Moreover, our analysis provides a molecular understanding of experimentally observed hidden allostery of PSD-95. We show that removing the distal third alpha helix from the binding site alters the allosteric pathway and decreases the binding affinity. Overall, these results indicate that (i) dynamics plays a key role in allosteric regulations of PDZs, (ii) the local changes in the residue interactions can lead to significant changes in the dynamics of allosteric regulations, and (iii) this might be the mechanism that each PDZ uses to tailor their binding specificities regulation.     

Conserved tertiary couplings stabilize elements in the PDZ fold,leading to characteristic patterns of domain conformational flexibility

Single‐domain allostery has been postulated to occur through intramolecular pathways of signaling within a protein structure. We had previously investigated these pathways by introducing a local thermal perturbation and analyzed the anisotropic propagation of structural changes throughout the protein. Here, we develop an improved approach, the Rotamerically Induced Perturbation (RIP), that identifies strong couplings between residues by analyzing the pathways of heat‐flow resulting from thermal excitation of rotameric rotations at individual residues. To explore the nature of these couplings, we calculate the complete coupling maps of 5 different PDZ domains. Although the PDZ domain is a well conserved structural fold that serves as a scaffold in many protein–protein complexes, different PDZ domains display unique patterns of conformational flexibility in response to ligand binding: some show a significant shift in a set of α‐helices, while others do not. Analysis of the coupling maps suggests a simple relationship between the computed couplings and observed conformational flexibility. In domains where the α‐helices are rigid, we find couplings of the α‐helices to the body of the protein, whereas in domains having ligand‐responsive α‐helices, no couplings are found. This leads to a model where the α‐helices are intrinsically dynamic but can be damped if sidechains interact at key tertiary contacts. These tertiary contacts correlate to high covariation contacts as identified by the statistical coupling analysis method. As these dynamic modules are exploited by various allosteric mechanisms, these tertiary contacts have been conserved by evolution.     

The interaction of Jagged‐1 cytoplasmic tail with afadin PDZ domain is local,folding‐independent,and tuned by phosphorylation

Jagged‐1, one of the five Notch ligands in man, is a membrane‐spanning protein made of a large extracellular region and a 125‐residue cytoplasmic tail bearing a C‐terminal PDZ recognition motif (¹²¹³RMEYIV¹²¹⁸). Binding of Jagged‐1 intracellular region to the PDZ domain of afadin, a protein located at cell–cell adherens junctions, couples Notch signaling with the adhesion system and the cytoskeleton. Using NMR chemical shift perturbation and surface plasmon resonance, we studied the interaction between the PDZ domain of afadin (AF6_PDZ) and a series of polypeptides comprising the PDZ‐binding motif. Chemical shift mapping of AF6_PDZ upon binding of ligands of different length (6, 24, and 133 residues) showed that the interaction is strictly local and involves only the binding groove in the PDZ. The recombinant protein corresponding to the entire intracellular region of Jagged‐1, J1_ic, is mainly disordered in solution, and chemical shift mapping of J1_ic in the presence of AF6_PDZ showed that binding is not coupled to folding. Binding studies on a series of 24‐residue peptides phosphorylated at different positions showed that phosphorylation of the tyrosine at position ‐2 of the PDZ‐binding motif decreases its affinity for AF6_PDZ, and may play a role in the modulation of this interaction. Finally, we show that the R1213Q mutation located in the PDZ‐binding motif and associated with extrahepatic biliary atresia increases the affinity for AF6_PDZ, suggesting that this syndrome may arise from an imbalance in the coupling of Notch signaling to the cytoskeleton. Copyright © 2010 John Wiley & Sons, Ltd.     

The Tiam1 PDZ Domain Couples to Syndecan1 and Promotes Cell-Matrix Adhesion

The T-cell lymphoma invasion and metastasis gene 1 (Tiam1) is a guanine exchange factor (GEF) for the Rho-family GTPase Rac1 that is crucial for the integrity of adherens junctions, tight junctions, and cell-matrix interactions. This GEF contains several protein-protein interaction domains, including a PDZ domain. Earlier studies identified a consensus PDZ-binding motif and a synthetic peptide capable of binding to the Tiam1 PDZ domain, but little is known about its ligand specificity and physiological role in cells. Here, we investigated the structure, specificity, and function of the Tiam1 PDZ domain. We determined the crystal structures of the Tiam1 PDZ domain free and in complex with a “model” peptide, which revealed the structural basis for ligand specificity. Protein database searches using the consensus PDZ-binding motif identified two eukaryotic cell adhesion proteins, Syndecan1 and Caspr4, as potential Tiam1 PDZ domain binding proteins. Equilibrium binding experiments confirmed that C-terminal peptides derived from Syndecan1 and Caspr4 bound the Tiam1 PDZ domain. NMR chemical shift perturbation experiments indicated that the Tiam1 PDZ/Syndecan1 and PDZ/Caspr4 complexes were structurally distinct and identified key residues likely to be responsible for ligand selectivity. Moreover, cell biological analysis established that Syndecan1 is a physiological binding partner of Tiam1 and that the PDZ domain has a function in cell-matrix adhesion and cell migration. Collectively, our data provide insight into the structure, specificity, and function of the Tiam1 PDZ domain. Importantly, our data report on a physiological role for the Tiam1 PDZ domain and establish a novel link between two previously unrelated signal transduction pathways, both of which are implicated in cancer.     

Unusual binding interactions in PDZ domain crystal structures help explain binding mechanisms

PDZ domains most commonly bind the C‐terminus of their protein targets. Typically the C‐terminal four residues of the protein target are considered as the binding motif, particularly the C‐terminal residue (P0) and third‐last residue (P‐2) that form the major contacts with the PDZ domain's “binding groove”. We solved crystal structures of seven human PDZ domains, including five of the seven PDLIM family members. The structures of GRASP, PDLIM2, PDLIM5, and PDLIM7 show a binding mode with only the C‐terminal P0 residue bound in the binding groove. Importantly, in some cases, the P‐2 residue formed interactions outside of the binding groove, providing insight into the influence of residues remote from the binding groove on selectivity. In the GRASP structure, we observed both canonical and noncanonical binding in the two molecules present in the asymmetric unit making a direct comparison of these binding modes possible. In addition, structures of the PDZ domains from PDLIM1 and PDLIM4 also presented here allow comparison with canonical binding for the PDLIM PDZ domain family. Although influenced by crystal packing arrangements, the structures nevertheless show that changes in the positions of PDZ domain side‐chains and the αB helix allow noncanonical binding interactions. These interactions may be indicative of intermediate states between unbound and fully bound PDZ domain and target protein. The noncanonical “perpendicular” binding observed potentially represents the general form of a kinetic intermediate. Comparison with canonical binding suggests that the rearrangement during binding involves both the PDZ domain and its ligand.     

Correlation analysis for protein evolutionary family based on amino acid position mutations and application in PDZ domain

Background

It has been widely recognized that the mutations at specific directions are caused by the functional constraints in protein family and the directional mutations at certain positions control the evolutionary direction of the protein family. The mutations at different positions, even distantly separated, are mutually coupled and form an evolutionary network. Finding the controlling mutative positions and the mutative network among residues are firstly important for protein rational design and enzyme engineering.

Methodology

A computational approach, namely amino acid position conservation-mutation correlation analysis (CMCA), is developed to predict mutually mutative positions and find the evolutionary network in protein family. The amino acid position mutative function, which is the foundational equation of CMCA measuring the mutation of a residue at a position, is derived from the MSA (multiple structure alignment) database of protein evolutionary family. Then the position conservation correlation matrix and position mutation correlation matrix is constructed from the amino acid position mutative equation. Unlike traditional SCA (statistical coupling analysis) approach, which is based on the statistical analysis of position conservations, the CMCA focuses on the correlation analysis of position mutations.

Conclusions

As an example the CMCA approach is used to study the PDZ domain of protein family, and the results well illustrate the distantly allosteric mechanism in PDZ protein family, and find the functional mutative network among residues. We expect that the CMCA approach may find applications in protein engineering study, and suggest new strategy to improve bioactivities and physicochemical properties of enzymes.     

Mutual information analysis of mutation,nonlinearity, and triple interactions in proteins

Mutations are the cause of several diseases as well as the underlying force of evolution. A thorough understanding of their biophysical consequences is essential. We present a computational framework for evaluating different levels of mutual information (MI) and its dependence on mutation. We used molecular dynamics trajectories of the third PDZ domain and its different mutations. Nonlinear MI between all residue pairs are calculated by tensor Hermite polynomials up to the fifth order and compared with results from multivariate Gaussian distribution of joint probabilities. We show that MI is written as the sum of a Gaussian and a nonlinear component. Results for the PDZ domain show that the Gaussian term gives a sufficiently accurate representation of MI when compared with nonlinear terms up to the fifth order. Changes in MI between residue pairs show the characteristic patterns resulting from specific mutations. Emergence of new peaks in the MI versus residue index plots of mutated PDZ shows how mutation may change allosteric pathways. Triple correlations are characterized by evaluating MI between triplets of residues. We observed that certain triplets are strongly affected by mutation. Susceptibility of residues to perturbation is obtained by MI and discussed in terms of linear response theory.     

Biophysical characterization of the complex between human papillomavirus E6 protein and synapse-associated protein 97

The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ(2) domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.     

Sequence requirement and subtype specificity in the high‐affinity interaction between human frizzled and dishevelled proteins

Members of the Wnt family of lipoglycoproteins initiate signaling by binding to Frizzled (Fz) receptors, and the signal is then relayed by Disheveled (Dvl). The Dvl PDZ domain is known to interact directly with a peptide derived from the KTXXXW motif of Fz7, which is conserved in all known Fz subtypes. We found that an extended region spanning the KTXXXW motif on both its N‐terminal and C‐terminal sides dramatically influences the affinity of peptides derived from Fz7 for Dvl PDZ. An alanine scanning study identified the specific residues external to the KTXXXW motif that are important for high‐affinity binding. In a circular dichroism analysis, mutation of some of these critical residues resulted in peptide conformational changes, suggesting that the secondary structure of the peptides contributes to Fz‐Dvl PDZ binding. Of the 10 known Fz subtypes, peptides derived from only Fz1, Fz2, Fz3, Fz4, and Fz7 directly bound to Dvl PDZ domain in our study. Other Fz subtypes, including some known to be involved in Wnt/β‐catenin signaling (Fz5, Fz9), did not bind to Dvl, suggesting that direct interaction with Dvl PDZ does not determine the subtype‐specific functionality of Fz. Molecular modeling and circular dichroism studies indicated that the Fz peptides that bind to Dvl PDZ domain form specific conformations that are different from those of nonbinding peptides.     

Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding

The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.     

Solution structure of HtrA PDZ domain from Streptococcus pneumoniae and its interaction with YYF-COOH containing peptides

High-temperature requirement A (HtrA), a highly conserved family of serine protease, plays crucial roles in protein quality control in prokaryotes and eukaryotes. The HtrA protein contains a C-terminal PDZ domain that mediates the proteolytic activity. Here we reported the solution structure of the HtrA PDZ domain from Streptococcus pneumoniae by NMR spectroscopy. Our results showed that the structure of HtrA PDZ domain, which contains three α-helices and five β-strands, illustrates conservation within the canonical PDZ domains. In addition, we demonstrated the interactions between S. pneumoniae HtrA PDZ domain and peptides with the motif XXX–YYF–COOH by surface plasmon resonance. Besides, we identified the ligand binding surface and the critical residues responsible for ligand binding of HtrA PDZ domain by chemical shift perturbation and site-directed mutagenesis.     

Distinct ligand specificity of the Tiam1 and Tiam2 PDZ domains

Guanine nucleotide exchange factor proteins of the Tiam family are activators of the Rho GTPase Rac1 and critical for cell morphology, adhesion, migration, and polarity. These proteins are modular and contain a variety of interaction domains, including a single post-synaptic density-95/discs large/zonula occludens-1 (PDZ) domain. Previous studies suggest that the specificities of the Tiam1 and Tiam2 PDZ domains are distinct. Here, we sought to conclusively define these specificities and determine their molecular origin. Using a combinatorial peptide library, we identified a consensus binding sequence for each PDZ domain. Analysis of these consensus sequences and binding assays with peptides derived from native proteins indicated that these two PDZ domains have overlapping but distinct specificities. We also identified residues in two regions (S(0) and S(-2) pockets) of the Tiam1 PDZ domain that are important determinants of ligand specificity. Site-directed mutagenesis of four nonconserved residues in these two regions along with peptide binding analyses confirmed that these residues are crucial for ligand affinity and specificity. Furthermore, double mutant cycle analysis of each region revealed energetic couplings that were dependent on the ligand being investigated. Remarkably, a Tiam1 PDZ domain quadruple mutant had the same specificity as the Tiam2 PDZ domain. Finally, analysis of Tiam family PDZ domain sequences indicated that the PDZ domains segregate into four distinct families based on the residues studied here. Collectively, our data suggest that Tiam family proteins have highly evolved PDZ domain-ligand interfaces with distinct specificities and that they have disparate PDZ domain-dependent biological functions.     

The interaction of PTP-BL PDZ domains with RIL: An enigmatic role for the RIL LIM domain

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signaling complexes. PDZ domains specifically bind short carboxyl-terminal peptides and occasionally internal sequences that structurally resemble peptide termini. Previously, using yeast two-hybrid methodology, we studied the interaction of two PDZ domains present in the large submembranous protein tyrosine phosphatase PTP-BL with the C-terminal half of the LIM domain-containing protein RIL. Deletion of the extreme RIL C-terminus did not eliminate binding, suggesting the presence of a PDZ binding site within the RIL LIM moiety. We have now performed experiments in mammalian cell lysates and found that the RIL C-terminus proper, but not the RIL LIM domain, can interact with PTP-BL, albeit very weakly. However, this interaction with PTP-BL PDZ domains is greatly enhanced when the combined RIL LIM domain and C-terminus is used, pointing to synergistic effects. NMR titration experiments and site-directed mutagenesis indicate that this result is not dependent on specific interactions that require surface exposed residues on the RIL LIM domain, suggesting a stabilizing role in the association with PTP-BL.     

Mechanisms for allosteric activation of protease DegS by ligand binding and oligomerization as revealed from molecular dynamics simulations

A local perturbation of a protein may lead to functional changes at some distal site, a phenomenon denoted as allostery. Here, we study the allosteric control of a protease using molecular dynamics simulations. The system considered is the bacterial protein DegS which includes a protease domain activated on ligand binding to an adjacent PDZ domain. Starting from crystallographic structures of DegS homo‐trimers, we perform simulations of the ligand‐free and ‐bound state of DegS at equilibrium. Considering a single protomer only, the trimeric state was mimicked by applying restraints on the residues in contact with other protomers in the DegS trimer. In addition, the bound state was also simulated without any restraints to mimic the monomer. Our results suggest that not only ligand release but also disassembly of a DegS trimer inhibits proteolytic activity. Considering various observables for structural changes, we infer allosteric pathways from the interface with other protomers to the active site. Moreover, we study how ligand release leads to (i) catalytically relevant changes involving residues 199–201 and (ii) a transition from a stretched to a bent conformation for residues 217–219 (which prohibits proper substrate binding). Finally, based on ligand‐induced C_α shifts we identify residues in contact with other protomers in the DegS trimer that likely transduce the perturbation from ligand release from a given protomer to adjacent protomers. These residues likely play a key role in the experimentally known effect of ligand release from a protomer on the proteolytic activity of the other protomers. Proteins 2016; 84:1690–1705. © 2016 Wiley Periodicals, Inc.     

The interaction of PTP-BL PDZ domains with RIL: an enigmatic role for the RIL LIM domain

PDZ domains are protein-protein interaction modules that are crucial for the assembly of structural and signaling complexes. PDZ domains specifically bind short carboxyl-terminal peptides and occasionally internal sequences that structurally resemble peptide termini. Previously, using yeast two-hybrid methodology, we studied the interaction of two PDZ domains present in the large submembranous protein tyrosine phosphatase PTP-BL with' the C-terminal half of the LIM domain-containing protein RIL. Deletion of the extreme RIL C-terminus did not eliminate binding, suggesting the presence of a PDZ binding site within the RIL LIM moiety. We have now performed experiments in mammalian cell lysates and found that the RIL C-terminus proper, but not the RIL LIM domain, can interact with PTP-BL, albeit very weakly. However, this interaction with PTP-BL PDZ domains is greatly enhanced when the combined RIL LIM domain and C-terminus is used, pointing to synergistic effects. NMR titration experiments and site-directed mutagenesis indicate that this result is not dependent on specific interactions that require surface exposed residues on the RIL LIM domain, suggesting a stabilizing role in the association with PTP-BL.     

Solution structure of GOPC PDZ domain and its interaction with the C-terminal motif of neuroligin

GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein) represents a PDZ domain-containing protein associated with the Golgi apparatus, which plays important roles in vesicular trafficking in secretory and endocytic pathways. GOPC interacts with many other proteins, such as the Wnt receptors Frizzled 8 and neuroligin via its PDZ domain. Neuroligin is a neural cell-adhesion molecule of the post-synapse, which binds to the presynapse molecule neurexin to form a heterotypic intercellular junction. Here we report the solution structure of the GOPC PDZ domain by NMR. Our results show that it is a canonical class I PDZ domain, which contains two alpha-helices and six beta-strands. Using chemical shift perturbation experiments, we further studied the binding properties of the GOPC PDZ domain with the C-terminal motif of neuroligin. The observations showed that the ensemble of the interaction belongs to fast exchange with low affinity. The 3D model of the GOPC PDZ domain/neuroligin C-terminal peptide complex was constructed with the aid of the molecular dynamics simulation method. Our discoveries provide insight into the specific interaction of the GOPC PDZ domain with the C-terminal peptide of Nlg and also provide a general insight about the possible binding mode of the interaction of Nlg with other PDZ domain-containing proteins.     

Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein.

The B class cell-attached ephrins mediate contact-dependent cell-cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified: one contains the PSD-95/Dlg/ZO-1 (PDZ) domain (for example PDZ-RGS3), and the second the Src homology 2 (SH2) domain (e.g. the Grb4 adaptor protein). The interaction with Grb4 requires phosphorylation of tyrosine residues on the conserved cytoplasmic C-terminal region of B ephrins, while binding to the PDZ domain is independent of tyrosine phosphorylation. However, the exact phosphorylation site(s) required for signaling remained obscure and it is also unknown whether the two classes of effectors can bind to B ephrins simultaneously or if the binding of one affects the binding of the other. We report here that phosphorylation of Tyr304 in the functional C-terminal region (residues 301-333) of ephrin B2 confers high-affinity binding to the SH2 domain of the Grb4 protein. Tyrosine phosphorylation at other candidate sites resulted in only minor change of the binding of Tyr304-phosphorylated ephrin B peptide (i.e. ephrinB2(301-333)-pY304) with the SH2 domain. (1)H-(15)N NMR HSQC experiments show that only the ephrinB2(301-333)-pY304 peptide forms a stable and specific binding complex with the SH2 domain of Grb4. The SH2 and PDZ domains were found to bind to the Tyr304 phosphopeptide both independently and at the same time, forming a three-component molecular complex. Taken together, our studies identify a novel SH2 domain binding motif, PHpY304EKV, on the cytoplasmic domains of B ephrins that may be essential for reverse signaling via the Grb4 adaptor protein alone or in concert with proteins containing PDZ domains.     

Regional covariation and its application for predicting protein contact patches

Correlated mutation analysis (CMA) is an effective approach for predicting functional and structural residue interactions from multiple sequence alignments (MSAs) of proteins. As nearby residues may also play a role in a given functional interaction, we were interested in seeing whether covarying sites were clustered, and whether this could be used to enhance the predictive power of CMA. A large‐scale search for coevolving regions within protein domains revealed that if two sites in a MSA covary, then neighboring sites in the alignment also typically covary, resulting in clusters of covarying residues. The program PatchD( http://www.uhnres.utoronto.ca/labs/tillier/ ) was developed to measure the covariation between disconnected sequence clusters to reveal patch covariation. Patches that exhibit strong covariation identify multiple residues that are generally nearby in the protein structure, suggesting that the detection of covarying patches can be used in conjunction with traditional CMA approaches to reveal functional interaction partners. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Some (dis)assembly required: partial unfolding in the Par-6 allosteric switch

Allostery is commonly described as a functional connection between two distant sites in a protein, where a binding event at one site alters affinity at the other. Here, we review the conformational dynamics that encode an allosteric switch in the PDZ domain of Par-6, which is a scaffold protein that organizes other proteins into a complex required to initiate and maintain cell polarity. NMR measurements revealed that the PDZ domain samples an evolutionarily conserved unfolding intermediate allowing rearrangement of two adjacent loop residues that control ligand binding affinity. Cdc42 binding to Par-6 creates a novel interface between the PDZ domain and the adjoining CRIB motif that stabilizes the high-affinity PDZ conformation. Thermodynamic and kinetic studies suggest that partial PDZ unfolding is an integral part of the Par-6 switching mechanism. The Par-6 CRIB-PDZ module illustrates two important structural aspects of protein evolution: the interface between adjacent domains in the same protein can give rise to allosteric regulation, and thermodynamic stability may be sacrificed to increase the sampling frequency of an unfolding intermediate required for conformational switching.     

Structural Basis for the Interaction between the Golgi Reassembly-stacking Protein GRASP65 and the Golgi Matrix Protein GM130

GM130 and GRASP65 are Golgi peripheral membrane proteins that play a key role in Golgi stacking and vesicle tethering. However, the molecular details of their interaction and their structural role as a functional unit remain unclear. Here, we present the crystal structure of the PDZ domains of GRASP65 in complex with the GM130 C-terminal peptide at 1.96-Å resolution. In contrast to previous findings proposing that GM130 interacts with GRASP65 at the PDZ2 domain only, our crystal structure of the complex indicates that GM130 binds to GRASP65 at two distinct sites concurrently and that both the PDZ1 and PDZ2 domains of GRASP65 participate in this molecular interaction. Mutagenesis experiments support these structural observations and demonstrate that they are required for GRASP65-GM130 association.