Localization and characterization of a heparin binding domain peptide of human von Willebrand factor.

Human von Willebrand factor, a plasma glycoprotein which plays a critical role in regulating hemostasis, binds heparin, but the physiological importance and mode of this interaction is poorly understood. Using the motif of an amino acid sequence of a consensus heparin binding synthetic peptide, a 23-residue sequence (Tyr565-Ala587) of human von Willebrand factor was identified that retains the consensus motif and binds heparin with affinity comparable with native von Willebrand factor and the consensus peptide. In a fluid phase binding assay, the Tyr565-Ala587 peptide competed effectively with von Willebrand factor for binding heparin. Synthesis and testing of peptides overlapping Tyr565-Ala587, as well as adjacent cationic regions, showed this core sequence to be the optimal linear binding domain. Far ultraviolet circular dichroism spectrometry of the Tyr565-Ala587 peptide suggested that the peptide undergoes conformational change upon binding heparin. The Tyr565-Ala587 peptide thus encompasses part (or all) of a functionally important heparin binding domain of von Willebrand factor. Further study of this and related peptides may be useful for exploring how heparin may influence von Willebrand factor-mediated platelet hemostasis.     

Solution structure of the phosphotyrosine binding (PTB) domain of human tensin2 protein in complex with deleted in liver cancer 1 (DLC1) peptide reveals a novel peptide binding mode

The protein deleted in liver cancer 1 (DLC1) interacts with the tensin family of focal adhesion proteins to play a role as a tumor suppressor in a wide spectrum of human cancers. This interaction has been proven to be crucial to the oncogenic inhibitory capacity and focal adhesion localization of DLC1. The phosphotyrosine binding (PTB) domain of tensin2 predominantly interacts with a novel site on DLC1, not the canonical NPXY motif. In this study, we characterized this interaction biochemically and determined the complex structure of tensin2 PTB domain with DLC1 peptide by NMR spectroscopy. Our HADDOCK-derived complex structure model elucidates the molecular mechanism by which tensin2 PTB domain recognizes DLC1 peptide and reveals a PTB-peptide binding mode that is unique in that peptide occupies the binding site opposite to the canonical NPXY motif interaction site with the peptide utilizing a non-canonical binding motif to bind in an extended conformation and that the N-terminal helix, which is unique to some Shc- and Dab-like PTB domains, is required for binding. Mutations of crucial residues defined for the PTB-DLC1 interaction affected the co-localization of DLC1 and tensin2 in cells and abolished DLC1-mediated growth suppression of hepatocellular carcinoma cells. This tensin2 PTB-DLC1 peptide complex with a novel binding mode extends the versatile binding repertoire of the PTB domains in mediating diverse cellular signaling pathways as well as provides a molecular and structural basis for better understanding the tumor-suppressive activity of DLC1 and tensin2.     

A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.

The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.     

The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure.

We have determined the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of stretches of A.T-rich DNA. Three highly conserved regions within each of the known HMG-I proteins is closely related to the consensus sequence T-P-K-R-P-R-G-R-P-K-K. A synthetic oligopeptide corresponding to this consensus "binding domain" (BD) sequence specifically binds to substrate DNA in a manner similar to the intact HMG-I proteins. Molecular Corey-Pauling-Koltun model building and computer simulations employing energy minimization programs to predict structure suggest that the consensus BD peptide has a secondary structure similar to the antitumor and antiviral drugs netropsin and distamycin and to the dye Hoechst 33258. In vitro these ligands, which also preferentially bind to A.T-rich DNA, have been demonstrated to effectively compete with both the BD peptide and the HMG-I proteins for DNA binding. The BD peptide also contains novel structural features such as a predicted Asx bend or "hook" at its amino-terminal end and laterally projecting cationic Arg/Lys side chains or "bristles" which may contribute to the binding properties of the HMG-I proteins. The predicted BD peptide structure, which we refer to as the "A.T-hook," represents a previously undescribed DNA-binding motif capable of binding to the minor groove of stretches of A.T base pairs.     

Evolution of binding affinity in a WW domain probed by phage display

The WW domain is an approximately 38 residue peptide-binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one-third of its three-stranded antiparallel beta-sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high-affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild-type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.     

Prediction of binding affinities between the human amphiphysin-1 SH3 domain and its peptide ligands using homology modeling, molecular dynamics and molecular field analysis

The SH3 domain of the human protein amphiphysin-1, which plays important roles in clathrin-mediated endocytosis, actin function and signaling transduction, can recognize peptide motif PXRPXR (X is any amino acid) with high affinity and specificity. We have constructed a complex structure of the amphiphysin-1 SH3 domain and a high-affinity peptide ligand PLPRRPPRA using homology modeling and molecular docking, which was optimized by molecular dynamics (MD). Three-dimensional quantitative structure-affinity relationship (3D-QSAR) analyses on the 200 peptides with known binding affinities to the amphiphysin-1 SH3 domain was then performed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The best CoMSIA model showed promising predictive power, giving good predictions for about 95% of the peptides in the test set (absolute prediction errors less than 1.0). It was used to validate peptide-SH3 binding structure and provide insight into the structural requirements for binding of peptides to SH3 domains. Finally, MD simulations were performed to analyze the interaction between the SH3 domain and another peptide GFPRRPPPRG that contains with the PXRPXsR (s represents residues with small side chains) motif. MD simulations demonstrated that the binding conformation of GFPRRPPPRG is quite different from that of PLPRRPPRAA especially the four residues at the C terminal, which may explain why the CoMSIA model cannot give good predictions on the peptides of the PXRPXsR motif. Because of its efficiency and predictive power, the 3D-QSAR model can be used as a scoring filter for predicting peptide sequences bound to SH3 domains.     

Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins

Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca(2+) coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 μM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+) binding with a K(D) of 12 μM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.     

Structural basis for specific binding of the Gads SH3 domain to an RxxK motif-containing SLP-76 peptide: a novel mode of peptide recognition

The SH3 domain, which normally recognizes proline-rich sequences, has the potential to bind motifs with an RxxK consensus. To explore this novel specificity, we have determined the solution structure of the Gads T cell adaptor C-terminal SH3 domain in complex with an RSTK-containing peptide, representing its physiological binding site on the SLP-76 docking protein. The SLP-76 peptide engages four distinct binding pockets on the surface of the Gads SH3 domain and upon binding adopts a unique structure characterized by a right-handed 3(10) helix at the RSTK locus, in contrast to the left-handed polyproline type II helix formed by canonical proline-rich SH3 ligands. The structure, and supporting mutagenesis and peptide binding data, reveal a novel mode of ligand recognition by SH3 domains.     

Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.     

Diversity in the SH2 domain family phosphotyrosyl peptide binding site

Src homology 2 (SH2) domains are approximately 100 residue phosphotyrosyl peptide binding modules found in signalling proteins and are important targets for therapeutic intervention. The peptide binding site is evolutionarily well conserved, particularly at the two major binding pockets, pTyr and pTyr + 3. We present a computational analysis of diversity within the peptide binding region and discuss molecular recognition beyond the conventional binding motif, drawing attention to novel conserved ligand interaction sites which may be exploitable in ligand binding studies. The peptide binding site is defined by selecting crystal contacts and domains are clustered according to binding site residue similarity. Comparison with a classification based on experimental peptide screening reveals a high level of qualitative agreement, indicating that the method is able independently to generate functional information. A conservation scoring method reveals extensive patches of conservation in some groups not present across the whole family, challenging the notion that the domains recognise only a linear phosphopeptide sequence. Conservation difference maps determine group-dependent clusters of conserved residues that are not seen when considering a larger experimentally determined group. Many of these residues contact the peptide outside the pTyr to pTyr + 3 motif, challenging the conventional view that this motif is largely responsible for ligand recognition and discrimination.     

Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional Gbeta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy

The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000) Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser(879) and Ser(880), are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gbeta-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade.     

Structure of the C-terminal phosphotyrosine interaction domain of Fe65L1 complexed with the cytoplasmic tail of amyloid precursor protein reveals a novel peptide binding mode

Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I beta-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel beta-sheet with the beta5 strand of the PID domain, the binding mode typically observed in the PID/PTB.peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn alpha-helix and interacts extensively with the C-terminal alpha-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB.peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins.     

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.     

Crystal structure of the molecular chaperone HscA substrate binding domain complexed with the IscU recognition peptide ELPPVKIHC

HscA, a specialized bacterial Hsp70-class molecular chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif. We report the crystal structure of the substrate-binding domain of HscA (SBD, residues 389-616) from Escherichia coli bound to an IscU-derived peptide, ELPPVKIHC. The crystals belong to the space group I222 and contain a single molecule in the asymmetric unit. Molecular replacement with the E.coli DnaK(SBD) model was used for phasing, and the HscA(SBD)-peptide model was refined to Rfactor=17.4% (Rfree=21.0%) at 1.95 A resolution. The overall structure of HscA(SBD) is similar to that of DnaK(SBD), although the alpha-helical subdomain (residues 506-613) is shifted up to 10 A relative to the beta-sandwich subdomain (residues 389-498) when compared to DnaK(SBD). The ELPPVKIHC peptide is bound in an extended conformation in a hydrophobic cleft in the beta-subdomain, which appears to be solvent-accessible via a narrow passageway between the alpha and beta-subdomains. The bound peptide is positioned in the reverse orientation of that observed in the DnaK(SBD)-NRLLLTG peptide complex placing the N and C termini of the peptide on opposite sides of the HscA(SBD) relative to the DnaK(SBD) complex. Modeling of the peptide in the DnaK-like forward orientation suggests that differences in hydrogen bonding interactions in the binding cleft and electrostatic interactions involving surface residues near the cleft contribute to the observed directional preference.     

Characterization of a synthetic peptide corresponding to a receptor binding domain of mouse interferon gamma.

A receptor binding region of mouse interferon gamma (IFN gamma) has previously been localized to the N-terminal 39 amino acids of the molecule by use of synthetic peptides and monoclonal antibodies. In this report, a detailed analysis of the synthetic peptide corresponding to this region, IFN gamma (1-39), is presented. Circular dichroism (CD) spectroscopy indicated that the peptide has stable secondary structure under aqueous conditions and adopts a combination of alpha-helical and random structure. A peptide lacking two N-terminal amino acids, IFN gamma (3-39), had similar secondary structure and equivalent ability to compete for receptor binding, while peptides lacking four or more N-terminal residues had reduced alpha-helical structure and did not inhibit 125I-IFN gamma binding. Substitution of proline, a helix-destabilizing amino acid, for leucine (residue 8) of a predicted amphipathic alpha-helix (residues 3-12), IFN gamma (1-39) [Pro]8, resulted in a substantial reduction in the helical content of the peptide, supporting the presence of helical structure in this region. However, destabilization of the helix did not reduce the competitive ability of the peptide. A peptide lacking eight C-terminal residues, IFN gamma (1-31), did not block 125I-IFN gamma binding and had no detectable alpha-helical structure, suggesting a requirement of the predicted second alpha-helix (residues 20-34) for receptor interaction and helix stabilization. Substitution of phenylalanine for tyrosine at position 14, IFN gamma (1-39) [Phe]14, a central location of a predicted omega-loop structure, did not affect the secondary structure associated with the region yet resulted in a 30-fold increase in receptor competition.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR solution structure of the focal adhesion targeting domain of focal adhesion kinase in complex with a paxillin LD peptide: evidence for a two-site binding model

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is regulated by integrins. Upon activation, FAK generates signals that modulate crucial cell functions, including cell proliferation, migration, and survival. The C-terminal focal adhesion targeting (FAT) sequence mediates localization of FAK to discrete regions in the cell called focal adhesions. Several binding partners for the FAT domain of FAK have been identified, including paxillin. We have determined the solution structure of the avian FAT domain in complex with a peptide mimicking the LD2 motif of paxillin by NMR spectroscopy. The FAT domain retains a similar fold to that found in the unliganded form when complexed to the paxillin-derived LD2 peptide, an antiparallel four-helix bundle. However, noticeable conformational changes were observed upon the LD2 peptide binding, especially the position of helix 4. Multiple lines of evidence, including the results obtained from isothermal titration calorimetry, intermolecular nuclear Overhauser effects, mutagenesis, and protection from paramagnetic line broadening, support the existence of two distinct paxillin-binding sites on the opposite faces of the FAT domain. The structure of the FAT domain-LD2 complex was modeled using the program HADDOCK based on our solution structure of the LD2-bound FAT domain and mutagenesis data. Our model of the FAT domain-LD2 complex provides insight into the molecular basis of FAK-paxillin binding interactions, which will aid in understanding the role of paxillin in FAK targeting and signaling.     

Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9

Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently. We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 beta-strands, which form two large twisted anti-parallel beta-sheets folding into a beta-sandwich. Three short alpha-helices were also identified. The beta-strands are linked by several loops and turns. These structural features of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops. Screening of a peptide library [XXX(pT)XXX] against FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K(d)=4-70 microM). These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53. The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously.     

Insulin-regulated aminopeptidase: analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine beta-naphthylamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.