Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor.

The Dedicator Of CytoKinesis (DOCK) family of atypical guanine nucleotide exchange factors activates the Rho family GTPases Rac and/or Cdc42 through DOCK homology region 2 (DHR-2). Previous structural analyses of the DHR-2 domains of DOCK2 and DOCK9 have shown that they preferentially bind Rac1 and Cdc42, respectively; however, the molecular mechanism by which DHR-2 distinguishes between these GTPases is unclear. Here we report the crystal structure of the Cdc42-bound form of the DOCK7 DHR-2 domain showing dual specificity for Rac1 and Cdc42. The structure revealed increased substrate tolerance of DOCK7 at the interfaces with switch 1 and residue 56 of Cdc42. Furthermore, molecular dynamics simulations showed a closed-to-open conformational change in the DOCK7 DHR-2 domain between the Cdc42- and Rac1-bound states by lobe B displacement. Our results suggest that lobe B acts as a sensor for identifying different switch 1 conformations and explain how DOCK7 recognizes both Rac1 and Cdc42.

Structural Basis for the Inhibition of Cyclin G-Associated Kinase by Gefitinib.

Gefitinib is the molecular target drug for advanced non-small-cell lung cancer. The primary target of gefitinib is the positive mutation of epidermal growth factor receptor, but it also inhibits cyclin G-associated kinase (GAK). To reveal the molecular bases of GAK and gefitinib binding, structure analyses were conducted and determined two forms of the gefitinib-bound nanobody⋅GAK kinase domain complex structures. The first form, GAK_1, has one gefitinib at the ATP binding pocket, whereas the second form, GAK_2, binds one each in the ATP binding site and a novel binding site adjacent to the activation segment C-terminal helix, a unique element of the Numb-associated kinase family. In the novel binding site, gefitinib binds in the hydrophobic groove around the activation segment, disrupting the conserved hydrogen bonds for the catalytic activity. These structures suggest possibilities for the development of selective GAK inhibitors for viral infections, such as the hepatitis C virus.

Phosphorylated and non-phosphorylated HCK kinase domains produced by cell-free protein expression.

Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant proteins easily. However, especially in the case of protein kinase, product solubility remains one of the major challenges. To overcome this problem, we prepared a chaperone-supplemented extract from Escherichia coli BL21 cells harboring a plasmid encoding a set of chaperone genes, dnaK, dnaJ, and grpE. We explored cell-disruption procedures and constructed an efficient protein synthesis system. Employing this system, we produced the kinase domain of human hematopoietic cell kinase (HCK) to obtain further structural information about its molecular interaction with one of its inhibitors, previously developed by our group (RK-20449). Lower reaction temperature improved the solubility, and addition of a protein phosphatase (YpoH) facilitated the homogeneous production of the non-phosphorylated kinase domain. Crystals of the purified product were obtained and the kinase-inhibitor complex structure was solved at 1.7 Šresolution. In addition, results of kinase activity measurement, using a synthetic substrate, showed that the kinase activity was facilitated by autophosphorylation at Tyr416, as confirmed by the peptide mass mapping.

Thioether Macrocyclic Peptides Selected against TET1 Compact Catalytic Domain Inhibit TET1 Catalytic Activity.

The ten-eleven translocation (TET) protein family, consisting of three isoforms (TET1/2/3), have been found in mammalian cells and have a crucial role in 5-methylcytosine demethylation in genomic DNA through the catalysis of oxidation reactions assisted by 2-oxoglutarate (2OG). DNA methylation/demethylation contributes to the regulation of gene expression at the transcriptional level, and recent studies have revealed that TET1 is highly elevated in malignant cells of various diseases and related to malignant alteration. TET1 inhibitors based on a scaffold of thioether macrocyclic peptides, which have been discovered by the random nonstandard peptide integrated discovery (RaPID) system, are reported. The affinity-based selection was performed against the TET1 compact catalytic domain (TET1CCD) to yield thioether macrocyclic peptides. These peptides exhibited inhibitory activity of the TET1 catalytic domain (TET1CD), with an IC50 value as low as 1.1 µm. One of the peptides, TiP1, was also able to inhibit TET1CD over TET2CD with tenfold selectivity, although it was likely to target the 2OG binding site; this provides a good starting point to develop more selective inhibitors.

Interhelical interactions between D92 and C218 in the cytoplasmic domain regulate proton uptake upon N-decay in the proton transport of Acetabularia rhodopsin II.

Acetabularia rhodopsin II (ARII or Ace2), an outward light-driven algal proton pump found in the giant unicellular marine alga Acetabularia acetabulum, has a unique property in the cytoplasmic (CP) side of its channel. The X-ray crystal structure of ARII in a dark state suggested the formation of an interhelical hydrogen bond between C218ARII and D92ARII, an internal proton donor to the Schiff base (Wada et al., 2011). In this report, we investigated the photocycles of two mutants at position C218ARII: C218AARII which disrupts the interaction with D92ARII, and C218SARII which potentially forms a stronger hydrogen bond. Both mutants exhibited slower photocycles compared to the wild-type pump. Together with several kinetic changes of the photoproducts in the first half of the photocycle, these replacements led to specific retardation of the N-to-O transition in the second half of the photocycle. In addition, measurements of the flash-induced proton uptake and release using a pH-sensitive indium-tin oxide electrode revealed a concomitant delay in the proton uptake. These observations strongly suggest the importance of a native weak hydrogen bond between C218ARII and D92ARII for proper proton translocation in the CP channel during N-decay. A putative role for the D92ARII-C218ARII interhelical hydrogen bond in the function of ARII is discussed.

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates.

Central catalytic domain of BRAP (RNF52) recognizes the types of ubiquitin chains and utilizes oligo-ubiquitin for ubiquitylation.

Really interesting new gene (RING)-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein (BRCA1)-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/ß plait (NBP), really interesting new gene (RING) zinc finger, ubiquitin-specific protease (UBP)-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated. In the present study, we discovered that the RNF52 domain, comprising NBP-RING-ZfUBP-CC, binds to ubiquitin chains (oligo-ubiquitin) but not to the ubiquitin monomers, and can utilize various ubiquitin chains for ubiquitylation and auto-ubiquitylation. The RNF52 domain preferentially bound to M1- and K63-linked di-ubiquitin chains, weakly to K27-linked chains, but not to K6-, K11-, or K48-linked chains. The binding preferences of the RNF52 domain for ubiquitin-linkage types corresponded to ubiquitin usage in the ubiquitylation reaction, except for K11-, K29-, and K33-linked chains. Additionally, the RNF52 domain directly ligated the intact M1-linked, tri-, and tetra-ubiquitin chains and recognized the structural alterations caused by the phosphomimetic mutation of these ubiquitin chains. Full-length BRAP had nearly the same specificity for the ubiquitin-chain types as the RNF52 domain alone. Mass spectrometry analysis of oligomeric ubiquitylation products, mediated by the RNF52 domain, revealed that the ubiquitin-linkage types and auto-ubiquitylation sites depend on the length of ubiquitin chains. Here, we propose a model for the oligomeric ubiquitylation process, controlled by the RNF52 domain, which is not a sequential assembly process involving monomers.

Parallel homodimer structures of the extracellular domains of the voltage-gated sodium channel ß4 subunit explain its role in cell-cell adhesion.

Voltage-gated sodium channels (VGSCs) are transmembrane proteins required for the generation of action potentials in excitable cells and essential for propagating electrical impulses along nerve cells. VGSCs are complexes of a pore-forming α subunit and auxiliary ß subunits, designated as ß1/ß1B-ß4 (encoded by SCN1B-4B, respectively), which also function in cell-cell adhesion. We previously reported the structural basis for the trans homophilic interaction of the ß4 subunit, which contributes to its adhesive function. Here, using crystallographic and biochemical analyses, we show that the ß4 extracellular domains directly interact with each other in a parallel manner that involves an intermolecular disulfide bond between the unpaired Cys residues (Cys58) in the loop connecting strands B and C and intermolecular hydrophobic and hydrogen-bonding interactions of the N-terminal segments (Ser30-Val35). Under reducing conditions, an N-terminally deleted ß4 mutant exhibited decreased cell adhesion compared with the wild type, indicating that the ß4 cis dimer contributes to the trans homophilic interaction of ß4 in cell-cell adhesion. Furthermore, this mutant exhibited increased association with the α subunit, indicating that the cis dimerization of ß4 affects α-ß4 complex formation. These observations provide the structural basis for the parallel dimer formation of ß4 in VGSCs and reveal its mechanism in cell-cell adhesion.

Structural basis of cucumisin protease activity regulation by its propeptide.

Cucumisin [EC 3.4.21.25], a subtilisin-like serine endopeptidase, was isolated from melon fruit, Cucumis melo L. Mature cucumisin (67 kDa, 621 residues) is produced by removal of the propeptide (10 kDa, 88 residues) from the cucumisin precursor by subsequence processing. It is reported that cucumisin is inhibited by its own propeptide. The crystal structure of mature cucumisin is reported to be composed of three domains: the subtilisin-like catalytic domain, the protease-associated domain and the C-terminal fibronectin-III-like domain. In this study, the crystal structure of the mature cucumisin•propeptide complex was determined by the molecular replacement method and refined at 1.95 Å resolution. In this complex, the propeptide had a domain of the α-ß sandwich motif with four-stranded antiparallel ß-sheets, two helices and a strand of the C-terminal region. The ß-sheets of the propeptide bind to two parallel surface helices of cucumisin through hydrophobic interaction and 27 hydrogen bonds. The C-terminus of the propeptide binds to the cleft of the active site as peptide substrates. The inhibitory assay suggested that the C-terminal seven residues of the propeptide do not inhibit the cucumisin activity. The crystal structure of the cucumisin•propeptide complex revealed the regulation mechanism of cucumisin activity.

Structural basis for disruption of claudin assembly in tight junctions by an enterotoxin.

The food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~35 kDa) that specifically targets human claudin-4, among the 26 human claudin proteins, and causes diarrhea by fluid accumulation in the intestinal cavity. The C-terminal domain of the Clostridium perfringens enterotoxin (C-CPE, ~15 kDa) binds tightly to claudin-4, and disrupts the intestinal tight junction barriers. In this study, we determined the 3.5-Šresolution crystal structure of the cell-free synthesized human claudin-4•C-CPE complex, which is significantly different from the structure of the off-target complex of an engineered C-CPE with mouse claudin-19. The claudin-4•C-CPE complex structure demonstrated the mechanism underlying claudin assembly disruption. A comparison of the present C-CPE-bound structure of claudin-4 with the enterotoxin-free claudin-15 structure revealed sophisticated C-CPE-induced conformation changes of the extracellular segments, induced on the foundation of the rigid four-transmembrane-helix bundle structure. These conformation changes provide a mechanistic model for the disruption of the lateral assembly of claudin molecules. Furthermore, the present novel structural mechanism for selecting a specific member of the claudin family can be used as the foundation to develop novel medically important technologies to selectively regulate the tight junctions formed by claudin family members in different organs.

Structural Mechanism for Light-driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3.

The light-driven inward chloride ion-pumping rhodopsin Nonlabens marinus rhodopsin-3 (NM-R3), from a marine flavobacterium, belongs to a phylogenetic lineage distinct from the halorhodopsins known as archaeal inward chloride ion-pumping rhodopsins. NM-R3 and halorhodopsin have distinct motif sequences that are important for chloride ion binding and transport. In this study, we present the crystal structure of a new type of light-driven chloride ion pump, NM-R3, at 1.58 Å resolution. The structure revealed the chloride ion translocation pathway and showed that a single chloride ion resides near the Schiff base. The overall structure, chloride ion-binding site, and translocation pathway of NM-R3 are different from those of halorhodopsin. Unexpectedly, this NM-R3 structure is similar to the crystal structure of the light-driven outward sodium ion pump, Krokinobacter eikastus rhodopsin 2. Structural and mutational analyses of NM-R3 revealed that most of the important amino acid residues for chloride ion pumping exist in the ion influx region, located on the extracellular side of NM-R3. In contrast, on the opposite side, the cytoplasmic regions of K. eikastus rhodopsin 2 were reportedly important for sodium ion pumping. These results provide new insight into ion selection mechanisms in ion pumping rhodopsins, in which the ion influx regions of both the inward and outward pumps are important for their ion selectivities.

Cell-free methods to produce structurally intact mammalian membrane proteins.

The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin.

Structure-based site-directed photo-crosslinking analyses of multimeric cell-adhesive interactions of voltage-gated sodium channel ß subunits.

The ß1, ß2, and ß4 subunits of voltage-gated sodium channels reportedly function as cell adhesion molecules. The present crystallographic analysis of the ß4 extracellular domain revealed an antiparallel arrangement of the ß4 molecules in the crystal lattice. The interface between the two antiparallel ß4 molecules is asymmetric, and results in a multimeric assembly. Structure-based mutagenesis and site-directed photo-crosslinking analyses of the ß4-mediated cell-cell adhesion revealed that the interface between the antiparallel ß4 molecules corresponds to that in the trans homophilic interaction for the multimeric assembly of ß4 in cell-cell adhesion. This trans interaction mode is also employed in the ß1-mediated cell-cell adhesion. Moreover, the ß1 gene mutations associated with generalized epilepsy with febrile seizures plus (GEFS+) impaired the ß1-mediated cell-cell adhesion, which should underlie the GEFS+ pathogenesis. Thus, the structural basis for the ß-subunit-mediated cell-cell adhesion has been established.

Binding interactions of the peripheral stalk subunit isoforms from human V-ATPase.

The mammalian peripheral stalk subunits of the vacuolar-type H(+)-ATPases (V-ATPases) possess several isoforms (C1, C2, E1, E2, G1, G2, G3, a1, a2, a3, and a4), which may play significant role in regulating ATPase assembly and disassembly in different tissues. To better understand the structure and function of V-ATPase, we expressed and purified several isoforms of the human V-ATPase peripheral stalk: E1G1, E1G2, E1G3, E2G1, E2G2, E2G3, C1, C2, H, a1NT, and a2NT. Here, we investigated and characterized the isoforms of the peripheral stalk region of human V-ATPase with respect to their affinity and kinetics in different combination. We found that different isoforms interacted in a similar manner with the isoforms of other subunits. The differences in binding affinities among isoforms were minor from our in vitro studies. However, such minor differences from the binding interaction among isoforms might provide valuable information for the future structural-functional studies of this holoenzyme.

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum.

Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.

Crystal structures of the human adiponectin receptors.

Adiponectin stimulation of its receptors, AdipoR1 and AdipoR2, increases the activities of 5' AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR), respectively, thereby contributing to healthy longevity as key anti-diabetic molecules. AdipoR1 and AdipoR2 were predicted to contain seven transmembrane helices with the opposite topology to G-protein-coupled receptors. Here we report the crystal structures of human AdipoR1 and AdipoR2 at 2.9 and 2.4 Å resolution, respectively, which represent a novel class of receptor structure. The seven-transmembrane helices, conformationally distinct from those of G-protein-coupled receptors, enclose a large cavity where three conserved histidine residues coordinate a zinc ion. The zinc-binding structure may have a role in the adiponectin-stimulated AMPK phosphorylation and UCP2 upregulation. Adiponectin may broadly interact with the extracellular face, rather than the carboxy-terminal tail, of the receptors. The present information will facilitate the understanding of novel structure-function relationships and the development and optimization of AdipoR agonists for the treatment of obesity-related diseases, such as type 2 diabetes.

Expression, purification, crystallization, and preliminary X-ray crystallographic studies of the human adiponectin receptors, AdipoR1 and AdipoR2.

The adiponectin receptors (AdipoR1 and AdipoR2) are membrane proteins with seven transmembrane helices. These receptors regulate glucose and fatty acid metabolism, thereby ameliorating type 2 diabetes. The full-length human AdipoR1 and a series of N-terminally truncated mutants of human AdipoR1 and AdipoR2 were expressed in insect cells. In small-scale size exclusion chromatography, the truncated mutants AdipoR1Δ88 (residues 89-375) and AdipoR2Δ99 (residues 100-386) eluted mostly in the intact monodisperse state, while the others eluted primarily as aggregates. However, gel filtration chromatography of the large-scale preparation of the tag-affinity-purified AdipoR1Δ88 revealed the presence of an excessive amount of the aggregated state over the intact state. Since aggregation due to contaminating nucleic acids may have occurred during the sample concentration step, anion-exchange column chromatography was performed immediately after affinity chromatography, to separate the intact AdipoR1Δ88 from the aggregating species. The separated intact AdipoR1Δ88 did not undergo further aggregation, and was successfully purified to homogeneity by gel filtration chromatography. The purified AdipoR1Δ88 and AdipoR2Δ99 proteins were characterized by thermostability assays with 7-diethylamino-3-(4-maleimidophenyl)-4-methyl coumarin, thin layer chromatography of bound lipids, and surface plasmon resonance analysis of ligand binding, demonstrating their structural integrities. The AdipoR1Δ88 and AdipoR2Δ99 proteins were crystallized with the anti-AdipoR1 monoclonal antibody Fv fragment, by the lipidic mesophase method. X-ray diffraction data sets were obtained at resolutions of 2.8 and 2.4 Å, respectively.

Crystal structures of the S6K1 kinase domain in complexes with inhibitors.

Ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine protein kinase that plays an important role in the PIK3/mTOR signaling pathway, and is implicated in diseases including diabetes, obesity, and cancer. The crystal structures of the S6K1 kinase domain in complexes with staurosporine and the S6K1-specific inhibitor PF-4708671 have been reported. In the present study, five compounds (F108, F109, F176, F177, and F179) were newly identified by in silico screening of a chemical library and kinase assay. The crystal structures of the five inhibitors in complexes with the S6K1 kinase domain were determined at resolutions between 1.85 and 2.10 Å. All of the inhibitors bound to the ATP binding site, lying along the P-loop, while the activation loop stayed in the inactive form. Compound F179, with a carbonyl group in the middle of the molecule, altered the αC helix conformation by interacting with the invariant Lys123. Compounds F176 and F177 bound slightly distant from the hinge region, and their sulfoamide groups formed polar interactions with the protein. The structural features required for the specific binding of inhibitors are discussed.

The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation.

Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2-ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain.

Structural basis for the specific recognition of the major antigenic peptide from the Japanese cedar pollen allergen Cry j 1 by HLA-DP5.

The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen and was shown to react with immunoglobulin E antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with 10 hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5·pCry j 1 complex forms several hydrogen bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2·pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide-binding specificities.