Cutting Edge: Coding single nucleotide polymorphisms of endoplasmic reticulum aminopeptidase 1 can affect antigenic peptide generation in vitro by influencing basic enzymatic properties of the enzyme

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis-Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis-Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant K(i), further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.     

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: Competition kinetics as a tool for enzyme specificity investigation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate’s N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.     

Probing the S1 specificity pocket of the aminopeptidases that generate antigenic peptides

ERAP1 (endoplasmic reticulum aminopeptidase 1), ERAP2 and IRAP (insulin-regulated aminopeptidase) are three homologous enzymes that play critical roles in the generation of antigenic peptides. These aminopeptidases excise amino acids from N-terminally extended precursors of antigenic peptides in order to generate the correct length epitopes for binding on to MHC class I molecules. The specificity of these peptidases can affect antigenic peptide selection, but has not yet been investigated in detail. In the present study we utilized a collection of 82 fluorigenic substrates to define a detailed selectivity profile for each of the three enzymes and to probe structural and functional features of the S1 (primary specificity) pocket. Molecular modelling of the three S1 pockets reveals substrate-enzyme interactions that are critical determinants for specificity. The substrate selectivity profiles suggest that IRAP largely combines the S1 specificity of ERAP1 and ERAP2, consistent with its proposed biological function. IRAP, however, does not achieve this dual specificity by simply combining structural features of ERAP1 and ERAP2, but rather by an unique amino acid change at position 541. The results of the present study provide insights on antigenic peptide selection and may prove valuable in designing selective inhibitors or activity markers for this class of enzymes.     

Concerted In Vitro Trimming of Viral HLA-B27-Restricted Ligands by Human ERAP1 and ERAP2 Aminopeptidases

In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.     

Combined Effects of Ankylosing Spondylitis-associated ERAP1 Polymorphisms Outside the Catalytic and Peptide-binding Sites on the Processing of Natural HLA-B27 Ligands

ERAP1 polymorphism involving residues 528 and 575/725 is associated with ankylosing spondylitis among HLA-B27-positive individuals. We used four recombinant variants to address the combined effects of the K528R and D575N polymorphism on the processing of HLA-B27 ligands. The hydrolysis of a fluorogenic substrate, Arg-528/Asp-575 < Lys-528/Asp-575 < Arg-528/Asn-575 < Lys-528/Asn-575, indicated that the relative activity of variants carrying Arg-528 or Lys-528 depends on residue 575. Asp-575 conferred lower activity than Asn-575, but the difference depended on residue 528. The same hierarchy was observed with synthetic precursors of HLA-B27 ligands, but the effects were peptide-dependent. Sometimes the epitope yields were variant-specific at all times. For other peptides, concomitant generation and destruction led to similar epitope amounts with all the variants at long, but not at short, digestion times. The generation/destruction balance of two related HLA-B27 ligands was analyzed in vitro and in live cells. Their relative yields at long digestion times were comparable with those from HLA-B27-positive cells, suggesting that ERAP1 was a major determinant of the abundance of these peptides in vivo. The hydrolysis of fluorogenic and peptide substrates by an HLA-B27 ligand or a shorter peptide, respectively, was increasingly inhibited as a function of ERAP1 activity, indicating that residues 528 and 575 affect substrate inhibition of ERAP1 trimming. The significant and complex effects of co-occurring ERAP1 polymorphisms on multiple HLA-B27 ligands, and their potential to alter the immunological and pathogenetic features of HLA-B27 as a function of the ERAP1 context, explain the epistatic association of both molecules in ankylosing spondylitis.     

The crystal structure of human endoplasmic reticulum aminopeptidase 2 reveals the atomic basis for distinct roles in antigen processing

Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding to major histocompatibility class I molecules. We report here the first structure of ERAP2 determined at 3.08 ? by X-ray crystallography. On the basis of residual electron density, a lysine residue has been modeled in the active site of the enzyme; thus, the structure corresponds to an enzyme-product complex. The overall domain organization is highly similar to that of the recently determined structure of ERAP1 in its closed conformation. A large internal cavity adjacent to the catalytic site can accommodate large peptide substrates. The ERAP2 structure provides a structural explanation for the different peptide N-terminal specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. A noncrystallographic dimer observed may constitute a model for a proposed ERAP1-ERAP2 heterodimer. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property of their biological role.     

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

The emergence of total drug-resistant tuberculosis (TDR-TB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new antimycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 N-terminal residues, the other mutated residues are essential for catalysis. Variations in K_m values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.     

The internal sequence of the peptide-substrate determines its N-terminus trimming by ERAP1

Background

Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims N-terminally extended antigenic peptide precursors down to mature antigenic peptides for presentation by major histocompatibility complex (MHC) class I molecules. ERAP1 has unique properties for an aminopeptidase being able to trim peptides in vitro based on their length and the nature of their C-termini.

Methodology/Principal Findings

In an effort to better understand the molecular mechanism that ERAP1 uses to trim peptides, we systematically analyzed the enzyme''s substrate preferences using collections of peptide substrates. We discovered strong internal sequence preferences of peptide N-terminus trimming by ERAP1. Preferences were only found for positively charged or hydrophobic residues resulting to trimming rate changes by up to 100 fold for single residue substitutions and more than 40,000 fold for multiple residue substitutions for peptides with identical N-termini. Molecular modelling of ERAP1 revealed a large internal cavity that carries a strong negative electrostatic potential and is large enough to accommodate peptides adjacent to the enzyme''s active site. This model can readily account for the strong preference for positively charged side chains.

Conclusions/Significance

To our knowledge no other aminopeptidase has been described to have such strong preferences for internal residues so distal to the N-terminus. Overall, our findings indicate that the internal sequence of the peptide can affect its trimming by ERAP1 as much as the peptide''s length and C-terminus. We therefore propose that ERAP1 recognizes the full length of its peptide-substrate and not just the N- and C- termini. It is possible that ERAP1 trimming preferences influence the rate of generation and the composition of antigenic peptides in vivo.     

The acylaminoacyl peptidase from Aeropyrum pernix K1 thought to be an exopeptidase displays endopeptidase activity

Mammalian acylaminoacyl peptidase, a member of the prolyl oligopeptidase family of serine peptidases, is an exopeptidase, which removes acylated amino acid residues from the N terminus of oligopeptides. We have investigated the kinetics and inhibitor binding of the orthologous acylaminoacyl peptidase from the thermophile Aeropyrum pernix K1 (ApAAP). Complex pH-rate profiles were found with charged substrates, indicating a strong electrostatic effect in the surroundings of the active site. Unexpectedly, we have found that oligopeptides can be hydrolysed beyond the N-terminal peptide bond, demonstrating that ApAAP exhibits endopeptidase activity. It was thought that the enzyme is specific for hydrophobic amino acids, in particular phenylalanine, in accord with the non-polar S1 subsite of ApAAP. However, cleavage after an Ala residue contradicted this notion and demonstrated that P1 residues of different nature may bind to the S1 subsite depending on the remaining peptide residues. The crystal structures of the complexes formed between the enzyme and product-like inhibitors identified the oxyanion-binding site unambiguously and demonstrated that the phenylalanine ring of the P1 peptide residue assumes a position different from that established in a previous study, using 4-nitrophenylphosphate. We have found that the substrate-binding site extends beyond the S2 subsite, being capable of binding peptides with a longer N terminus. The S2 subsite displays a non-polar character, which is unique among the enzymes of this family. The S3 site was identified as a hydrophobic region that does not form hydrogen bonds with the inhibitor P3 residue. The enzyme-inhibitor complexes revealed that, upon ligand-binding, the S1 subsite undergoes significant conformational changes, demonstrating the plasticity of the specificity site.     

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.     

Naturally processed HLA class II peptides reveal highly conserved immunogenic flanking region sequence preferences that reflect antigen processing rather than peptide-MHC interactions

MHC class II heterodimers bind peptides 12-20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRS: We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.     

Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence

The cDNAs corresponding to the mRNA encoding a polypeptide which is immunoreactive with the antisera specific to carcinoembryonic antigen (CEA) (1) are cloned. The amino acid sequences deduced from the nucleotide sequences of the cDNAs show that it is synthesized as a precursor with a signal peptide followed by 668 amino acids of the putative mature CEA peptide, whose N-terminal 24 amino acids and amino acids 286 to 295 exactly coincide with those known for N-terminal sequences of CEA (2) and NFA-1 (3), respectively. The first 108 N-terminal residues are followed by three very homologous repetitive domains of 178 residues each and then by 26 mostly hydrophobic residues which probably comprise a membrane anchor. Each repetitive domains contains 4 cysteines at precisely the same positions and as many as 28 possible N-glycosylation sites are found in the CEA peptide region agreeing with high carbohydrate content of purified CEA.     

Amino acid sequence of the signal peptide of mitochondrial nicotinamide nucleotide transhydrogenase as determined from the sequence of its messenger RNA

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase was recently deduced from isolated cDNAs and reported [Yamaguchi, M., Hatefi, Y., Trach, K., and Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767]. The cDNAs lacked the N-terminal coding region, however, and the 8 N-terminal residues were determined by protein sequencing. In the present study, the nucleotide sequence of the 5' upstream region was determined by dideoxynucleotide sequencing of the transhydrogenase messenger RNA, and amino acid sequences of the N-terminal region and the signal peptide of the enzyme were deduced from the nucleotide sequence. The N-terminal sequence of the enzyme as deduced from the mRNA sequence is the same as that determined by protein sequencing, with one difference. Protein sequencing showed Ser as the N-terminal residue. The mRNA sequence indicated that Ser is the second N-terminal residue, and the first is Cys. That preparations of the enzyme are mixtures of two polypeptides, one polypeptide being one residue shorter at the N terminus than the other, has been pointed out in the above reference. The signal peptide consists of 43 residues, is rich in basic (4 Lys, 2 Arg) and hydroxylated (4 Thr, 3 Ser) amino acids, and lacks acidic residues.     

NMR structural studies of the myristoylated N-terminus of ADP ribosylation factor 6 (Arf6)

Arf proteins are guanine nucleotide binding proteins that are implicated in endocytotic pathways and vesicle trafficking. The two widely studied isoforms of Arf proteins (Arf1 and Arf6) have different cellular functions and localizations but similar structures. Arf proteins have an N-terminal helix with a covalently bound myristoyl group. Except structural models, there are no three dimensional structures of the myristoylated N-terminal peptide or the intact myristoylated Arf proteins. However, understanding the role of both the myristoyl group and the N-terminal helix based on the details of their molecular structures is of great interest. In the solution structure of myristoylated N-terminal peptide of Arf6 described here, the myristoyl group folds toward the N-terminus to interact with the hydrophobic residues in particular, the phenyl ring. Also, the structure of the dodecylphosphocholine (DPC) micelle-bound of the peptide together with paramagnetic studies showed that the myristoyl group is inserted into the micelle while residues V4-G10 interact with the surface of the micelle. The structural differences between the unbound and micelle-bound myristoylated N-terminal peptide of Arf6 involves the myristoyl group and the side chains of the hydrophobic residues.     

Expression of endoplasmic reticulum aminopeptidases in EBV-B cell lines from healthy donors and in leukemia/lymphoma, carcinoma, and melanoma cell lines

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.     

Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides

The specificity of proteinase K from Tritirachium album Limber was determined using various synthetic peptide substrates. The esterase activity against N-acylated amino acid esters indicated that the enzyme is primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point. Secondary interaction for hydrolysis was also studied using peptide esters or others, which showed that the enzyme activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point. Thus, peptide chloromethyl ketone derivatives such as Cbz-Ala-Gly-PheCH₂Cl inactivated the enzyme activity markedly.     

Exploration of zinc-binding groups for the design of inhibitors for the oxytocinase subfamily of M1 aminopeptidases

The oxytocinase subfamily of M1 aminopeptidases consists of three members, ERAP1, ERAP2 and IRAP that play several important biological roles, including key functions in the generation of antigenic peptides that drive human immune responses. They represent emerging targets for pharmacological manipulation of the immune system, albeit lack of selective inhibitors is hampering these efforts. Most of the previously explored small-molecule binders target the active site of the enzymes via strong interactions with the catalytic zinc(II) atom and, while achieving increased potency, they suffer in selectivity. Continuing our earlier efforts on weaker zinc(II) binding groups (ZBG), like the 3,4-diaminobenzoic acid derivatives (DABA), we herein synthesized and biochemically evaluated analogues of nine potentially weak ZBGs, based on differential substitutions of functionalized pyridinone- and pyridinethione-scaffolds, nicotinic-, isonicotinic-, aminobenzoic- and hydrazinobenzoic-acids. Crystallographic analysis of two analogues in complex with a metalloprotease (MMP-12) revealed unexpected binding topologies, consistent with the observed affinities. Our results suggest that the potency of the compounds as inhibitors of ERAP1, ERAP2 and IRAP is primarily driven by the occupation of active-site specificity pockets and their proper orientation within the enzymes.     

Characterizing the N-terminal processing motif of MHC class I ligands

Most peptide ligands presented by MHC class I molecules are the product of an intracellular pathway comprising protein breakdown in the cytosol, transport into the endoplasmic reticulum, and successive N-terminal trimming events. The efficiency of each of these processes depends on the amino acid sequence of the presented ligand and its precursors. Thus, relating the amino acid composition N-terminal of presented ligands to the sequence specificity of processes in the pathway gives insight into the usage of ligand precursors in vivo. Examining the amino acid composition upstream the true N terminus of MHC class I ligands, we demonstrate the existence of a distinct N-terminal processing motif comprising approximately seven residues and matching the known preferences of proteasome and TAP, two key players in ligand processing. Furthermore, we find that some residues, which are preferred by both TAP and the proteasome, are underrepresented at positions immediately preceding the N terminus of MHC class I ligands. Based on experimentally determined aminopeptidase activities, this pattern suggests trimming next to the final N terminus to take place predominantly in the endoplasmic reticulum.     

Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair

Human centrin 2 is a component of the nucleotide excision repair system, as a subunit of the heterotrimer including xeroderma pigmentosum group C protein (XPC) and hHR23B. The C-terminal domain of centrin (C-HsCen2) binds strongly a peptide from the XPC protein (P1-XPC: N(847)-R(863)). Here, we characterize the solution Ca(2+)-dependent structural and molecular features of the C-HsCen2 in complex with P1-XPC, mainly using NMR spectroscopy and molecular modeling. The N-terminal half of the peptide, organized as an alpha helix is anchored into a deep hydrophobic cavity of the protein, because of three bulky hydrophobic residues in position 1-4-8 and electrostatic contacts with the centrin helix E. Investigation of the whole centrin interactions shows that the N-terminal domain of the protein is not involved in the complex formation and is structurally independent from the peptide-bound C-terminal domain. The complex may exist in three different binding conformations corresponding to zero, one, and two Ca(2+)-bound states, which may exchange with various rates and have distinct structural stability. The various features of the intermolecular interaction presented here constitute a centrin-specific mode for the target binding.     

Identification of a potential hydrophobic peptide binding site in the C-terminal arm of trigger factor

Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding, and the M-domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear, and the residues or regions directly involved in substrate binding have not yet been identified. Here, a hydrophobic probe, bis-ANS, was used to characterize potential substrate-binding regions. Results showed that bis-ANS binds TF with a 1:1 stoichiometry and a K(d) of 16 microM, and it can be covalently incorporated into TF by UV-light irradiation. A single bis-ANS-labeled peptide was obtained by tryptic digestion and identified by MALDI-TOF mass spectrometry as Asn391-Lys392. In silico docking analysis identified a single potential binding site for bis-ANS on the TF molecule, which is adjacent to this dipeptide and lies in the pocket formed by the C-terminal arms. The bis-ANS-labeled TF completely lost the ability to assist GAPDH or lysozyme refolding and showed increased protection toward cleavage by alpha-chymotrypsin, suggesting blocking of hydrophobic residues. The C-terminal truncation mutant TF389 also showed no chaperone activity and could not bind bis-ANS. These results suggest that bis-ANS binding may mimic binding of a substrate peptide and that the C-terminal region of TF plays an important role in hydrophobic binding and chaperone function.