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1.
H H?rig A C Young N J Papadopoulos T P DiLorenzo S G Nathenson 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(8):4434-4441
MHC class I molecules usually bind short peptides of 8-10 amino acids, and binding is dependent on allele-specific anchor residues. However, in a number of cellular systems, class I molecules have been found containing peptides longer than the canonical size. To understand the structural requirements for MHC binding of longer peptides, we used an in vitro class I MHC folding assay to examine peptide variants of the antigenic VSV 8 mer core peptide containing length extensions at either their N or C terminus. This approach allowed us to determine the ability of each peptide to productively form Kb/beta2-microglobulin/peptide complexes. We found that H-2Kb molecules can accommodate extended peptides, but only if the extension occurs at the C-terminal peptide end, and that hydrophobic flanking regions are preferred. Peptides extended at their N terminus did not promote productive formation of the trimolecular complex. A structural basis for such findings comes from molecular modeling of a H-2Kb/12 mer complex and comparative analysis of MHC class I structures. These analyses revealed that structural constraints in the A pocket of the class I peptide binding groove hinder the binding of N-terminal-extended peptides, whereas structural features at the C-terminal peptide residue pocket allow C-terminal peptide extensions to reach out of the cleft. These findings broaden our understanding of the inherent peptide binding and epitope selection criteria of the MHC class I molecule. Core peptides extended at their N terminus cannot bind, but peptide extensions at the C terminus are tolerated. 相似文献
2.
A set of combinatorial amphipathic helical peptides referred to as the KIA series has been screened to identify native-like helical bundles. The series contains the following consensus sequence: AKAxAAxxKAxAAxxKAGGY, where "x" positions are occupied by either Ala or Ile. The peptide sequences in the series comprise all possible combinations of four Ile residues occupying the six x positions. In each case, Ala occupied the two x positions not occupied by Ile. There are a total of 15 peptides in the KIA series; all of the peptides differ in the number of ridges and grooves formed by the Ile side chains, and two of the KIA peptides possess a canonical knobs-into-holes heptad repeat. The structure and stability of these 15 peptides and their pairwise mixtures were evaluated. One peptide in the series formed a stable four-helix bundle that folded with cooperativity similar to native proteins. Ten peptides assembled into molten globular helical assemblies, two peptides were unstructured, and two peptides assembled into helical filaments that were several micrometers long. One of the helical filament forming peptides could be diverted from forming filaments by the addition of another KIA peptide, and resulted in the formation of a heteromeric six-helix bundle. This study demonstrates that combinatorial peptides composed of only three types of amino acids can form a diverse array of structures, some of which are native-like. 相似文献
3.
Structural resiliency of an EGF-like subdomain bound to its target protein, thrombin. 总被引:2,自引:0,他引:2
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R. Hrabal E. A. Komives F. Ni 《Protein science : a publication of the Protein Society》1996,5(2):195-203
The thrombin-bound structures of native peptide fragments from the fifth EGF-like domain of thrombomodulin were determined by use of NMR and transferred NOE spectroscopy. The bound peptides assume an EGF-like structure of an antiparallel beta-sheet, a novel structural motif observed for a bound peptide in protein-peptide complexes. There is a remarkable structural resiliency of this structure motif manifested in its ability to accommodate a different number of residues within the disulfide loop. Docking experiments revealed that the key contacts with thrombin are hydrophobic interactions between the side chains of residues Ile 414 and Ile 424 of thrombomodulin and a hydrophobic pocket on the thrombin surface. Residues Leu 415, Phe 419, and Ile 420, which would have been buried in intact EGF-like domains, are unfavorably exposed in the complex of thrombin with the EGF-like thrombomodulin fragment, thus providing a rationale for the enhancement of binding affinity upon the deletion of Ile 420. The unique beta-sheet structures of the bound peptides are specified by the presence of disulfide bridges in the peptides because a corresponding linear thrombomodulin fragment folds into a sheet structure with a different backbone topology. The different bound conformations for the linear and the cyclized peptides indicate that side-chain interactions within a specific environment may dictate the folding of bound peptides in protein-peptide complexes. 相似文献
4.
Seema Jagota Jayakumar Rajadas 《International journal of peptide research and therapeutics》2012,18(1):53-61
Genetic, biochemical and pathological evidence support that self-assembly of amyloid-beta (Aβ) peptide into toxic aggregates
is implicated as the cause of Alzheimer’s disease. An attractive therapeutic strategy for the treatment of AD is to prevent
or interfere with Aβ aggregation. A systematic investigation of the effects of proline-, glycine-, arginine- and lysine- containing
peptides (PGKLVYA, KKLVFFARRRRA and KKLVFFA) on the beta-amyloid aggregation was made using FTIR, circular dichroism, ANS
binding, ThT binding and TEM analysis. These peptides are based on the central hydrophobic region of Aβ (residues 16–20),
which is believed to be crucial in Aβ self-association. There is increasing evidence to suggest that protein aggregation,
including amyloid fibril formation results from the strong self-association tendency of the partially folded intermediates.
Addition of PGKLVYA and KKLVFFARRRRA resulted in increase in ANS fluorescence intensity, suggesting enhanced exposure of hydrophobic
surface area. As observed by ThT and TEM analysis PGKLVYA and KKLVFFARRRRA promote non-fibrillar ensembles, while peptide
KKLVFFA accelerated the fibrillization of Aβ peptide by stabilizing intermolecular interactions. Circular dichroism and FTIR
data showed that PGKLVYA and KKLVFFARRRRA effectively prevented amyloid-beta (Aβ) peptide adopting the beta-sheet secondary
structure correlated with fibrillogenesis. This result indicates that PGKLVYA and KKLVFFARRRRA might have triggered another
mechanism of Aβ assembly. 相似文献
5.
The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1. 相似文献
6.
《Bioorganic chemistry》1987,15(2):183-193
1H and 13C NMR signal assignments for 8-anilinonaphthalenesulfonic acid (ANS) and dansyl amide (DNSA) are achieved using high-field spectra, decoupling, and two-dimensional NMR techniques as well as shift differences between conjugate acid and bases. Complexation of ANS and DNSA with a macrocyclic azoniacyclophane is measured by fluorescence and by NMR shift titration, furnishing an independent check for the equilibrium constant determination. The complexation-induced shifts (CIS) for ANS and DNSA are analyzed on the basis of aromatic ring current and linear electric field effect models. Comparison of equilibrium constants of the cyclophane and different substrates shows that, e.g., for ANS, lipophilic/hydrophobic binding dominates over electrostatic effects despite the presence of charges and the absence of a lipophilic cap or bottom on the receptor molecule. 相似文献
7.
The HIV-1 envelope glycoprotein gp41 fusion intermediate is a promising drug target for inhibiting viral entry. However, drug development has been impeded by challenges inherent in mediating the underlying protein–protein interaction. Here we report on the identification of fragments that bind to a C-terminal sub-pocket adjacent to the well-known hydrophobic pocket on the NHR coiled coil. Using a specifically designed assay and ligand-based NMR screening of a fragment library, we identified a thioenylaminopyrazole compound with a dissociation constant of ~500 μM. Interaction with the C-terminal sub-pocket was confirmed by paramagnetic relaxation enhancement NMR experiments, which also yielded the binding mode. Shape-based similarity searching detected additional phenylpyrazole and phenyltriazole fragments within the library, enriching the hit rate over random screening, and revealing molecular features required for activity. Discovery of the novel scaffolds and binding mechanism suggests avenues for extending the interaction surface and improving the potency of a hydrophobic pocket binding inhibitor. 相似文献
8.
《Journal of molecular biology》1996,257(2):398-411
The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an α-helix that lies across the β-sheet, with residues Ile93, Ile94 and Met97 making contacts with Leu44, Phe56 and Ile58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the β-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135° to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from Ile93 and Ile94 to Ile58, Leu 41, Val62 and His10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface. 相似文献
9.
应用圆二色性,内源荧光和疏水荧光探针法进一步研究Tl8肽的溶液构象及其相互转化。发现T18肽在水溶液中为β折叠结构,且在高浓度(>1mg/ml)时形成疏水聚合物;Lys15和Ile3-Ile4是形成β折叠疏水簇的关键因素。并讨论了蛋白质肽链和溶剂环境对肽段二级结构的调制作用。 相似文献
10.
Robert W. Griffith 《Origins of life and evolution of the biosphere》2009,39(6):517-531
Among various scenarios that attempt to explain how life arose, the RNA world is currently the most widely accepted scientific
hypothesis among biologists. However, the RNA world is logistically implausible and doesn’t explain how translation arose
and DNA became incorporated into living systems. Here I propose an alternative hypothesis for life’s origin based on cooperation
between simple nucleic acids, peptides and lipids. Organic matter that accumulated on the prebiotic Earth segregated into
phases in the ocean based on density and solubility. Synthesis of complex organic monomers and polymerization reactions occurred
within a surface hydrophilic layer and at its aqueous and atmospheric interfaces. Replication of nucleic acids and translation
of peptides began at the emulsified interface between hydrophobic and aqueous layers. At the core of the protobiont was a
family of short nucleic acids bearing arginine’s codon and anticodon that added this amino acid to pre-formed peptides. In
turn, the survival and replication of nucleic acid was aided by the peptides. The arginine-enriched peptides served to sequester
and transfer phosphate bond energy and acted as cohesive agents, aggregating nucleic acids and keeping them at the interface. 相似文献
11.
12.
Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers 总被引:6,自引:0,他引:6
Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles. Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment. Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations. The variants were moderately less active in vitro but more effective in animal models. Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL. All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes. Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents. None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis. As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold. In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E. coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability. The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells. Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes. Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia. 相似文献
13.
The four-helical protein Im7 folds via a rapidly formed on-pathway intermediate (k(UI)=3000 s(-1) at pH 7.0, 10 degrees C) that contains three (helices I, II and IV) of the four native alpha-helices. The relatively slow (k(IN)=300 s(-1)) conversion of this intermediate into the native structure is driven by the folding and docking of the six residue helix III onto the developing hydrophobic core. Here, we describe the structural properties of four Im7* variants designed to trap the protein in the intermediate state by disrupting the stabilising interactions formed between helix III and the rest of the protein structure. In two of these variants (I54A and L53AI54A), hydrophobic residues within helix III have been mutated to alanine, whilst in the other two mutants the sequence encompassing the native helix III was replaced by a glycine linker, three (H3G3) or six (H3G6) residues in length. All four variants were shown to be monomeric, as judged by analytical ultracentrifugation, and highly helical as measured by far-UV CD. In addition, all the variants denature co-operatively and have a stability (DeltaG(UF)) and buried hydrophobic surface area (M(UF)) similar to those of the on-pathway kinetic intermediate. Structural characterisation of these variants using 1-anilino-8-napthalene sulphonic acid (ANS) binding, near-UV CD and 1D (1)H NMR demonstrate further that the trapped intermediate ensemble is highly structured with little exposed hydrophobic surface area. Interestingly, however, the structural properties of the variants I54A and L53AI54A differ in detail from those of H3G3 and H3G6. In particular, the single tryptophan residue, located near the end of helix IV, and distant from helix III, is in a distinct environment in the two sets of mutants as judged by fluorescence, near-UV CD and the sensitivity of tryptophan fluorescence to iodide quenching. Overall, the results confirm previous kinetic analysis that demonstrated the hierarchical folding of Im7 via an on-pathway intermediate, and show that this species is a highly helical ensemble with a well-formed hydrophobic core. By contrast with the native state, however, the intermediate ensemble is flexible enough to change in response to mutation, its structural properties being tailored by residues in the sequence encompassing the native helix III. 相似文献
14.
Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency 总被引:1,自引:0,他引:1
Human angiogenin (Ang) is an RNase in the pancreatic RNase superfamily that induces angiogenesis. Its catalytic activity is comparatively weak, but nonetheless critical for biological activity. The crystal structure of Ang has shown that enzymatic potency is attenuated in part by the obstructive positioning of Gln117 within the B(1) pyrimidine binding pocket, and that the C-terminal segment of residues 117-123 must reorient for Ang to bind and cleave RNA. The native closed conformation appears to be stabilized by Gln117-Thr44 and Asp116-Ser118 hydrogen bonds, as well as hydrophobic packing of Ile119 and Phe120. Consistent with this view, Q117G, D116H, and I119A/F120A variants are 4-30-fold more active than Ang. Here we have determined crystal structures for these variants to examine the structural basis for the activity increases. In all three cases, the C-terminal segment remains obstructive, demonstrating that none of the residues that has been replaced is essential for maintaining the closed conformation. The Q117G structure shows no changes other than the loss of the side chain of residue 117, whereas those of D116H and I119A/F120A reveal C-terminal perturbations beyond the replacement site, suggesting that the native closed conformation has been destabilized. Thus, the interactions of Gln117 seem to be less important than those of residues 116, 119, and 120 for stabilization. In D116H, His116 does not replicate either of the hydrogen bonds of Asp116 with Ser118 and instead forms a water-mediated interaction with catalytic residue His114; residues 117-121 deviate significantly from their positions in Ang. In I119A/F120A, the segment of residues 117-123 has become highly mobile and all of the interactions thought to position Gln117 have been weakened or lost; the space occupied by Phe120 in Ang is partially filled by Arg101, which has moved several angstroms. A crystal structure was also determined for the deletion mutant des(121-123), which has 10-fold reduced activity toward large substrates. The structure is consistent with the earlier proposal that residues 121-123 form part of a peripheral substrate binding subsite, but also raises the possibility that changes in the position of another residue, Lys82, might be responsible for the decreased activity of this variant. 相似文献
15.
The interaction of calmodulin with fluorescent and photoreactive model peptides: evidence for a short interdomain separation 总被引:2,自引:0,他引:2
Calmodulin is known to bind target enzymes and basic, amphiphilic peptides in a Ca2(+)-dependent manner. Recently, we introduced a photoaffinity label, p-benzoylphenylalanine (Bpa), into the sequence of a model, alpha-helical, calmodulin-binding peptide. When the Bpa residue was introduced at the third position of the peptide, Met-144 on the C-terminal domain of calmodulin was labeled, whereas when the photolabel was placed at the thirteenth position, Met-71 on the N-terminal domain was labeled. Assuming that both peptides bind in similar orientations, these results are not consistent with the crystal structure of calmodulin, in which the domains are held at a significant distance from one another by a long alpha-helical segment. To test the assumption that both peptides bind in similar orientations, we have synthesized a calmodulin-binding peptide with the photolabel in both the third and the thirteenth positions. Upon photolysis, this peptide forms a cross-link between Met-71 and Met-124 on the N- and C-terminal domains, respectively. Furthermore, a peptide with a Bpa in the thirteenth position and a Trp residue in the third position was also synthesized. After photocross-linking the Bpa residue of this peptide to Met-71 of calmodulin, it could be shown that the fluorescence properties of the Trp residue were consistent with its side chain being buried in a hydrophobic pocket on the C-terminal domain of calmodulin. These data indicate that, when complexed with basic, amphiphilic peptides, calmodulin can adopt a conformation in which its two domains are significantly closer than in the crystal structure of the uncomplexed protein. 相似文献
16.
17.
High-affinity ligands of non-peptidic nature, binding to the class I major histocompatibility complex protein HLA B*2705 whose expression is strongly linked to the pathogenesis of the autoimmune disease ankylosing spondylitis, should give way to a selective immunotherapy by blocking or antagonising the interaction with autoreactive T cell clones. Here we present experimental data on the binding of modified peptides, designed to optimally bind to HLA-B*2705 by filling a hydrophobic binding pocket (pocket D) with nonencoded aromatic amino acids. Three peptides with altered side chains (alpha-naphthylalanine, beta-naphthylalanine and homophenylalanine) in position 3 were synthesised. The thermal denaturation profiles of the HLA protein in complex with the modified peptides, monitored by circular dichroism spectroscopy, showed a significant shift towards higher melting temperatures with respect to the parent T cell epitope. The proposed binding mode of the nonnatural peptides was checked by site-directed mutagenesis of the pocket D, hypothesised to accommodate the large hydrophobic side chains. Reducing the size and depth of the pocket by mutating Leu l56 into Trp only affects the binding of the non-natural ligands, thus providing experimental evidence that the nonnatural peptide amino acids bind as predicted to the host MHC protein. © 1998 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
18.
The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided. 相似文献
19.
The PDZ domain of neuronal nitric oxide synthase (nNOS) functions as a scaffold for organizing the signal transduction complex of the enzyme. The NMR structure of a complex composed of the nNOS PDZ domain and an associated peptide suggests that a two-stranded beta-sheet C-terminal to the canonical PDZ domain may mediate its interaction with the PDZ domains of postsynaptic density-95 and alpha-syntrophin. The structure also provides the molecular basis of recognition of Asp-X-Val-COOH peptides by the nNOS PDZ domain. The role of the C-terminal extension in Asp-X-Val-COOH peptide binding is investigated. Additionally, NMR studies further show that the Asp-X-Val-COOH peptide and a C-terminal peptide from a novel cytosolic protein named CAPON bind to the same pocket of the nNOS PDZ domain. 相似文献
20.
Wang Q Deloia MA Kang Y Litchke C Zhang N Titus MA Walters KJ 《Protein science : a publication of the Protein Society》2007,16(2):189-196
Myosins play essential roles in migration, cytokinesis, endocytosis, and adhesion. They are composed of a large N-terminal motor domain with ATPase and actin binding sites and C-terminal neck and tail regions, whose functional roles and structural context in the protein are less well characterized. The tail regions of myosins I, IV, VII, XII, and XV each contain a putative SH3 domain that may be involved in protein-protein interactions. SH3 domains are reported to bind proline-rich motifs, especially "PxxP" sequences, and such interactions serve regulatory functions. The activity of Src, PI3, and Itk kinases, for example, is regulated by intramolecular interactions between their SH3 domain and internal proline-rich sequences. Here, we use NMR spectroscopy to reveal the structure of a protein construct from Dictyostelium myosin VII (DdM7) spanning A1620-T1706, which contains its SH3 domain and adjacent proline-rich region. The SH3 domain forms the signature beta-barrel architecture found in other SH3 domains, with conserved tryptophan and tyrosine residues forming a hydrophobic pocket known to bind "PxxP" motifs. In addition, acidic residues in the RT or n-Src loops are available to interact with the basic anchoring residues that are typically found in ligands or proteins that bind SH3 domains. The DdM7 SH3 differs in the hydrophobicity of the second pocket formed by the 3(10) helix and following beta-strand, which contains polar rather than hydrophobic side chains. Most unusual, however, is that this domain binds its adjacent proline-rich region at a surface remote from the region previously identified to bind "PxxP" motifs. The interaction may affect the orientation of the tail without sacrificing the availability of the canonical "PxxP"-binding surface. 相似文献