Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.     

HLA-DM Mediates Epitope Selection by a “Compare-Exchange” Mechanism when a Potential Peptide Pool Is Available

Background

HLA-DM (DM) mediates exchange of peptides bound to MHC class II (MHCII) during the epitope selection process. Although DM has been shown to have two activities, peptide release and MHC class II refolding, a clear characterization of the mechanism by which DM facilitates peptide exchange has remained elusive.

Methodology/Principal Findings

We have previously demonstrated that peptide binding to and dissociation from MHCII in the absence of DM are cooperative processes, likely related to conformational changes in the peptide-MHCII complex. Here we show that DM promotes peptide release by a non-cooperative process, whereas it enhances cooperative folding of the exchange peptide. Through electron paramagnetic resonance (EPR) and fluorescence polarization (FP) we show that DM releases prebound peptide very poorly in the absence of a candidate peptide for the exchange process. The affinity and concentration of the candidate peptide are also important for the release of the prebound peptide. Increased fluorescence energy transfer between the prebound and exchange peptides in the presence of DM is evidence for a tetramolecular complex which resolves in favor of the peptide that has superior folding properties.

Conclusion/Significance

This study shows that both the peptide releasing activity on loaded MHCII and the facilitating of MHCII binding by a candidate exchange peptide are integral to DM mediated epitope selection. The exchange process is initiated only in the presence of candidate peptides, avoiding possible release of a prebound peptide and loss of a potential epitope. In a tetramolecular transitional complex, the candidate peptides are checked for their ability to replace the pre-bound peptide with a geometry that allows the rebinding of the original peptide. Thus, DM promotes a “compare-exchange” sorting algorithm on an available peptide pool. Such a “third party”-mediated mechanism may be generally applicable for diverse ligand recognition in other biological systems.     

Structural factors contributing to DM susceptibility of MHC class II/peptide complexes

Peptide loading of MHC class II (MHCII) molecules is assisted by HLA-DM, which releases invariant chain peptides from newly synthesized MHCII and edits the peptide repertoire. Determinants of susceptibility of peptide/MHCII complexes to DM remain controversial, however. Here we have measured peptide dissociation in the presence and the absence of DM for 36 different complexes of varying intrinsic stability. We found large variations in DM susceptibility for different complexes using either soluble or full-length HLA-DM. The DM effect was significantly less for unstable complexes than for stable ones, although this correlation was modest. Peptide sequence- and allele-dependent interactions along the entire length of the Ag binding groove influenced DM susceptibility. We also observed differences in DM susceptibility during peptide association. Thus, the peptide repertoire displayed to CD4(+) T cells is the result of a mechanistically complicated editing process and cannot be simply predicted from the intrinsic stability of the complexes in the absence of DM.     

Cutting edge: TCR contacts as anchors: effects on affinity and HLA-DM stability

Peptides presented via the class II MHC (MHCII) pathway are selected based on affinity for MHCII and stability in the presence of HLA-DM. Currently, epitope selection is thought to be controlled by the ability of peptide to sequester "anchor" residues into pockets in the MHCII. Residues exhibiting higher levels of solvent accessibility have been shown to contact TCR, but their roles in affinity and complex stability have not been directly studied. Using the HLA-DR1-binding influenza peptide, hemagglutinin (306-318), as a model, we show that side chain substitutions at these positions influence affinity and HLA-DM stability. Multiple substitutions reduce affinity to a greater extent than the loss of the major P1 anchor residue. We propose that these effects may be mediated through the H-bond network. These results demonstrate the importance of solvent-exposed residues in epitope selection and blur the distinctions between anchor and TCR contact residues.     

Enhanced catalytic action of HLA-DM on the exchange of peptides lacking backbone hydrogen bonds between their N-terminal region and the MHC class II alpha-chain

The class II MHC homolog HLA-DM catalyzes exchange of peptides bound to class II MHC proteins, and is an important component of the Ag presentation machinery. The mechanism of HLA-DM-mediated catalysis is largely obscure. HLA-DM catalyzes exchange of peptides of varying sequence, suggesting that a peptide sequence-independent component of the MHC-peptide interaction could be involved in the catalytic process. Twelve conserved hydrogen bonds between the peptide backbone and the MHC are a prominent sequence-independent feature of the MHC-peptide interaction. To evaluate the relative importance of these hydrogen bonds toward HLA-DM action, we prepared peptide variants that lacked the ability to form one or more of the hydrogen bonds as a result of backbone amide N-methylation or truncation, and tested their ability to be exchanged by HLA-DM. We found that disruption of hydrogen bonds involving HLA-DR1 residues alpha51-53, a short extended segment at the N terminus of the alpha subunit helical region, led to heightened HLA-DM catalytic efficacy. We propose that those bonds are disrupted in the MHC conformation recognized by HLA-DM to allow structural transitions in that area during DM-assisted peptide release. These results suggest that peptides or compounds that bind MHC but cannot form these interactions would be preferentially edited out by HLA-DM.     

Characteristics of carbohydrate antigen binding to the presentation protein HLA-DR

Zwitterionic polysaccharide antigens (ZPSs) were recently shown to activate T cells in a class II major histocompatibility complex (MHCII)-dependent fashion requiring antigen presenting cell (APC)-mediated oxidative processing although little is known about the mechanism or affinity of carbohydrate presentation (Cobb BA, Wang Q, Tzianabos AO, Kasper DL. 2004. Polysaccharide processing and presentation by the MHCII pathway. Cell. 117:677-687). A recent study showed that the helical conformation of ZPSs (Wang Y, Kalka-Moll WM, Roehrl MH, Kasper DL. 2000. Structural basis of the abscess-modulating polysaccharide A2 from Bacteroides fragilis. Proc Natl Acad Sci USA. 97:13478-13483; Choi YH, Roehrl MH, Kasper DL, Wang JY. 2002. A unique structural pattern shared by T-cell-activating and abscess-regulating zwitterionic polysaccharides. Biochemistry. 41:15144-15151) is closely linked with immunogenic activity (Tzianabos AO, Onderdonk AB, Rosner B, Cisneros RL, Kasper DL. 1993. Structural features of polysaccharides that induce intra-abdominal abscesses. Science. 262:416-419) and is stabilized by a zwitterionic charge motif (Kreisman LS, Friedman JH, Neaga A, Cobb BA. 2007. Structure and function relations with a T-cell-activating polysaccharide antigen using circular dichroism. Glycobiology. 17:46-55), suggesting a strong carbohydrate structure-function relationship. In this study, we show that PSA, the ZPS from Bacteroides fragilis, associates with MHCII at high affinity and 1:1 stoichiometry through a mechanism mirroring peptide presentation. Interestingly, PSA binding was mutually exclusive with common MHCII antigens and showed significant allelic differences in binding affinity. The antigen exchange factor HLA-DM that catalyzes peptide antigen association with MHCII also increased the rate of ZPS association and was required for APC presentation and ZPS-mediated T cell activation. Finally, the zwitterionic nature of these antigens was required only for MHCII binding, and not endocytosis, processing, or vesicular trafficking to MHCII-containing vesicles. This report is the first quantitative analysis of the binding mechanism of carbohydrate antigens with MHCII and leads to a novel model for nontraditional MHCII antigen presentation during bacterial infections.     

Cooperativity during the formation of peptide/MHC class II complexes

To generate an effective immune response, class II major histocompatibility complex molecules (MHCII) must present a diverse array of peptide ligands for recognition by T lymphocytes. Peptide/MHCII complexes are stabilized by hydrophobic anchoring of peptide side chains to pockets in the MHCII protein and the formation of hydrogen bonds to the peptide backbone. Many current models of peptide/MHCII association assume an additive and independent contribution of the interactions between major MHCII pockets and corresponding side chains in the peptide. However, significant conformational rearrangements occur in both the peptide and MHCII during binding. Therefore, we hypothesize that peptide binding to MHCII could be viewed as a folding process in which both molecules cooperate to produce the final conformation. To directly test this hypothesis, we adapt a serial mutagenesis strategy to study cooperativity in the interaction of the human MHCII HLA-DR1 and a peptide derived from influenza hemagglutinin. Substitutions in either the peptide or HLA-DR1 that are predicted to interfere with hydrogen bond formation show cooperative effects on complex stability and affinity. Substitution of a peptide side chain that provides a hydrophobic contact also contributes to the cooperative effect, suggesting a role for all energetic sources in the folding process. We propose that cooperativity throughout the peptide-binding groove reflects the folding of segments of the MHCII molecule into helices around the peptide with a concomitant folding of the peptide into a polyproline helix. The implications of cooperativity for peptide/MHCII structure and epitope selection are discussed.     

Formation of two peptide/MHC II isomers is catalyzed differentially by HLA-DM

Major histocompatability class II proteins are transmembrane alphabeta-heterodimers that present peptides to T-cells. MHC II may bind exogenous peptides directly at the cell surface. Alternatively, peptides derived from processing of endosomal protein may bind to MHC II in endosomal compartments. There, HLA-DM catalyzes the formation of peptide/MHC complexes, which are then transported to the cell surface. Here we report evidence that the peptide Ii CLIP 81-104 binds to DR*0404 in two alternate registries, whose dissociation rates, while kinetically indistinguishable at pH 5.3 and 37 degrees C, are kinetically resolved in the presence of HLA-DM. In one registry isomer, CLIP Met 91 is placed in the N-terminal P1 pocket of DR*0404, and peptide dissociation is readily catalyzed by HLA-DM. In a second proposed registry, likely with CLIP Leu 97 in the P1 pocket, the complex is substantially less sensitive to HLA-DM catalysis. Without HLA-DM, or at pH 7, the fraction of each isomer formed in solution is relatively insensitive to the duration of incubation with peptide. However, with HLA-DM, the fraction of the DM-insensitive isomer is dramatically influenced by peptide incubation time. The mechanism of isomer formation appears to be determined by the HLA-DM-modified relative association to the two registries, followed by HLA-DM-catalyzed dissociation of each isomer and rebinding, leading to a final isomer composition determined by these kinetic constants. Intramolecular isomer interconversion does not appear to be involved. The behavior of these complexes may provide a model for peptide editing by DM in endosomes.     

Flexibility of the MHC class II peptide binding cleft in the bound, partially filled, and empty states: a molecular dynamics simulation study

Major histocompatibility (MHC) Class II cell surface proteins present antigenic peptides to the immune system. Class II structures in complex with peptides but not in the absence of peptide are known. Comparative molecular dynamics (MD) simulations of a Class II protein (HLA-DR3) with and without CLIP (invariant chain-associated protein) peptide were performed starting from the CLIP-bound crystal structure. Depending on the protonation of acidic residues in the P6 peptide-binding pocket the simulations stayed overall close to the start structure. The simulations without CLIP showed larger conformational fluctuations especially of alpha-helices flanking the binding cleft. Largest fluctuations without CLIP were observed in a helical segment near the peptide C-terminus binding region matching a segment recognized by antibodies specific for empty Class II proteins. Simulations on a Val86Tyr mutation that fills the peptide N-terminus binding P1 pocket or of a complex with a CLIP fragment (dipeptide) bound to P1 showed an unexpected long range effect. In both simulations the mobility not only of P1 but also of the entire binding cleft was reduced compared to simulations without CLIP. It correlates with the experimental finding that the CLIP fragment binding to P1 is sufficient to prevent antibody recognition specific for the empty form at a site distant from P1. The results suggest a mechanism how a local binding event of small peptides or of an exchange factor near P1 may promote peptide binding and exchange through a long range stabilization of the whole binding cleft in a receptive (near bound) conformation.     

Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2

Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade.     

Small molecules that enhance the catalytic efficiency of HLA-DM

HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. Large lateral surfaces involved in the DM:HLA-DR (DR) interaction have been defined, but the mechanism of catalysis is not understood. In this study, we describe four small molecules that accelerate DM-catalyzed peptide exchange. Mechanistic studies demonstrate that these small molecules substantially enhance the catalytic efficiency of DM, indicating that they make the transition state of the DM:DR/peptide complex energetically more favorable. These compounds fall into two functional classes: two compounds are active only in the presence of DM, and binding data for one show a direct interaction with DM. The remaining two compounds have partial activity in the absence of DM, suggesting that they may act at the interface between DM and DR/peptide. A hydrophobic ridge in the DMbeta1 domain was implicated in the catalysis of peptide exchange because the activity of three of these enhancers was substantially reduced by point mutations in this area.     

Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.     

Functional analysis of tryptophans alpha 62 and beta 120 on HLA-DM.

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM alphaW62A,betaW120A (DM(W62A/W120A)) double mutant was expressed in HLA-DR(+) HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM(W62A/W120A) removes CLIP as efficiently as its wild-type counterpart. DM(W62A/W120A) was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations alphaW62A and betaW120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM(W62A/)W120A were co-immunoprecipitated at pH 7. We conclude that the alpha62 and beta120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.     

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction

Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein’s involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1β and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed ‘hydrocarbon chain-first’ mechanism for C1P uptake, in contrast to the ‘lipid polar headgroup-first’ uptake used by most lipid-transfer proteins.     

A conformational change in the human major histocompatibility complex protein HLA-DR1 induced by peptide binding

To investigate a conformational change accompanying peptide binding to class II MHC proteins, we probed the structure of a soluble version of the human class II MHC protein HLA-DR1 in empty and peptide-loaded forms. Peptide binding induced a large decrease in the effective radius of the protein as determined by gel filtration, dynamic light scattering, and analytical ultracentrifugation. It caused a substantial increase in the cooperativity of thermal denaturation and induced alterations in MHC polypeptide backbone structure as determined by circular dichroism. These changes suggest a condensation of the protein around the bound peptide. An antibody specific for beta58-69 preferentially bound the empty protein, indicating that the peptide-induced conformational change involves the beta-subunit helical region. The conformational change may have important implications for the mechanisms of intracellular antigen presentation pathways.     

Structure and function relations with a T-cell-activating polysaccharide antigen using circular dichroism

Studies centered on understanding how molecular structure affects biological function have historically focused on proteins. Circular dichroism (CD) is commonly used to analyze protein secondary structure, yet its application to other molecules is far less explored. In fact, little is known about how glycan conformation might affect function, likely because of a lack of tools for measuring dynamic structural changes of carbohydrates. In the present study, we developed a method based on CD to monitor conformational changes in the zwitterionic T-cell-activating glycoantigen polysaccharide A1 (PSA). We found that PSA helical structure produces a CD spectrum that is strikingly similar to proteins rich in alpha-helical content and is equally sensitive to nonpolar solvents. Like conventional T-cell-dependent proteins, PSA requires processing before major histocompatibility complex class II (MHCII) binding. CD spectra of PSA fragments of varying sizes indicated that fragments smaller than three repeating units lack helical content and are incapable of MHCII binding. Likewise, neutralization of charged groups in the repeating unit resulted in major conformational changes as measured by CD, which correlated with a lack of MHCII presentation. These data represent two significant findings: CD can be used to measure conformational changes in carbohydrates and the functional epitope from PSA is dependent on a specific conformation that is stabilized by adjacent repeating units and a zwitterionic charge motif. As a result, this work demonstrates that CD is a valuable tool for use in functional glycomics efforts that seek to align chemical and conformational structure with biological activity.     

Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the beta subunit designed to fill the P1 pocket from within the protein (Gly(86) to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.     

Caspase-3 binds diverse P4 residues in peptides as revealed by crystallography and structural modeling

Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9–2.6 Å. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.     

The kinetics of PDZ domain-ligand interactions and implications for the binding mechanism

PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A + B --> A.B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex.