Biomedical subjects

Feng Ni

Publications and source records attributed to Feng Ni.

At least 19 recordsLinked to original sources

PubMed2025

Interaction between toll-like receptor 4 polymorphism and abdominal obesity on ovarian cancer risk in Chinese women.

OBJECTIVES: the aim of this study was to evaluate the impact of TLR4 gene single nucleotide polymorphisms (SNPs) and additional TLR4 gene SNP- SNP and SNP- abdominal obesity (AO) interaction on ovarian cancer (OC) risk. METHODS: Generalized multifactor dimensionality reduction method were utilized to identify the most informative interactions between four SNPs in the TLR4 gene and abdominal obesity. Logistic regression was employed to investigate the association between 4 SNPs within TLR4 gene and OC risk, and additional SNP- SNP and gene- AO interaction on OC risk, ORs (95%CI) were calculated. RESULTS: The analysis of logistic regression indicated a markedly elevated risk of OC in individuals carrying either the rs4986790-G or rs11536889-C alleles in the TLR4 gene compared to those with the standard genetic variations, adjusted ORs (95%CI) were 1.61 (1.28-1.96) and 1.48 (1.09-1.91). GMDR analysis indicated a significant two-locus model (p = 0.018) involving rs4986790 and rs11536889, and a significant two-locus model (p = 0.001) involving rs4986790 and AO. Participants with rs4986790- AG/GG and rs11536889GC/ CC genotype has the highest OC risk, compared to participants with rs4986790-AA and rs11536889-GG genotype, OR (95%CI) = 2.58 (1.46-3.71), and abdominal obese participants with rs4986790- AG/GG genotype have the highest OC risk, compared to non- abdominal obese participants with rs4986790-AA genotype, OR (95%CI) = 3.17 (1.78-4.58). CONCLUSIONS: The findings suggested that TLR4 gene rs4986790 and rs11536889 polymorphisms were associated with increased OC risk. Significant interaction also existed between rs4986790 and AO, which means that the WC levels may influence the impact of rs4986790 on OC risk.

Adult↗

PubMed2006

Improving solubility and refolding efficiency of human V(H)s by a novel mutational approach.

The antibody V(H) domains of camelids tend to be soluble and to resist aggregation, in contrast to human V(H) domains. For immunotherapy, attempts have therefore been made to improve the properties of human V(H)s by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama V(H) domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human V(H), derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human V(H)s with good antigen binding activity and solubility. The engineered human V(H) domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.

Amino Acid Sequence↗

PubMed2006

Three-dimensional structure and ligand interactions of the low molecular weight protein tyrosine phosphatase from Campylobacter jejuni.

A putative low molecular weight protein tyrosine phosphatase (LMW-PTP) was identified in the genome sequence of the bacterial pathogen, Campylobacter jejuni. This novel gene, cj1258, has sequence homology with a distinctive class of phosphatases widely distributed among prokaryotes and eukaryotes. We report here the solution structure of Cj1258 established by high-resolution NMR spectroscopy using NOE-derived distance restraints, hydrogen bond data, and torsion angle restraints. The three-dimensional structure consists of a central four-stranded parallel beta-sheet flanked by five alpha-helices, revealing an overall structural topology similar to those of the eukaryotic LMW-PTPs, such as human HCPTP-A, bovine BPTP, and Saccharomyces cerevisiae LTP1, and to those of the bacterial LMW-PTPs MPtpA from Mycobacterium tuberculosis and YwlE from Bacillus subtilis. The active site of the enzyme is flexible in solution and readily adapts to the binding of ligands, such as the phosphate ion. An NMR-based screen was carried out against a number of potential inhibitors and activators, including phosphonomethylphenylalanine, derivatives of the cinnamic acid, 2-hydroxy-5-nitrobenzaldehyde, cinnamaldehyde, adenine, and hypoxanthine. Despite its bacterial origin, both the three-dimensional structure and ligand-binding properties of Cj1258 suggest that this novel phosphatase may have functional roles close to those of eukaryotic and mammalian tyrosine phosphatases. The three-dimensional structure along with mapping of small-molecule binding will be discussed in the context of developing high-affinity inhibitors of this novel LMW-PTP.

Bacterial Proteins↗

PubMed2005

A bivalent dissectional analysis of the high-affinity interactions between Cdc42 and the Cdc42/Rac interactive binding domains of signaling kinases in Candida albicans.

The small GTPase Cdc42, a member of the highly conserved Rho family of intracellular GTPases, communicates with downstream signaling proteins via high-affinity interactions with the consensus Cdc42/Rac interactive binding (CRIB) polypeptide sequence. Previous biochemical and structural studies show that the CRIB motif itself is insufficient for high-affinity binding to Cdc42 but requires the sequence segment C-terminal to the CRIB motif for enhanced affinity. In this study, we have investigated the high-affinity (K(d) in units of nanomolar) associations of two highly homologous extended CRIB domains (eCRIBs) from the PAK kinases, Cla4 and Cst20, with Cdc42 from Candida albicans. (1)H-(15)N NMR heteronuclear NOE data of the eCRIB polypeptides in complex with Candida Cdc42 (CaCdc42) indicate that both eCRIB peptides have approximately two binding loci for CaCdc42. When each of the two eCRIB peptides is dissected into two fragments, the N-terminal fragments containing the minimal CRIB motif (mCRIB), mCla4 and mCst20, have relatively high binding affinities with dissociation constants (K(d)) of 4.2 and 0.43 microM, respectively. On the other hand, the C-terminal fragments (cCRIB), cCla4 and cCst20, exhibit significantly lower affinities for their binding to CaCdc42. The cCla4 peptide binds to CaCdc42 with a sub-millimolar K(d) of 275 microM, and the cCst20 peptide shows an even lower binding affinity (K(d) = 1160 microM). Cross-titration experiments with the cognate fragments show that the binding affinity of cCst20 is enhanced approximately 5.5-fold (K(d) = 207 microM) in the presence of saturating amounts of mCst20, and vice versa. No such effect is observed for the binding of cCla4 and mCla4. These results suggest that the Cdc42-CRIB system can be represented by a "dual recognition" model for protein-protein interactions [Kleanthous, C., et al. (1998) Mol. Microbiol. 28, 227-233], following much the same mechanisms of multivalent molecular interactions [Song, J., and Ni, F. (1998) Biochem. Cell Biol. 76, 177-188; Mammen, M., et al. (1998) Angew Chem., Int. Ed. 37, 2754-2794]. The bivalent modeling of linked peptide fragments shows that the binding of eCla4 follows a simple additivity/avidity model, while binding of eCst20 appears to have a more complex mechanism involving cooperative effects. The differential binding mechanisms between closely related eCRIB polypeptides and CaCdc42 provide a new molecular basis for understanding kinase activation and for the design of antifungal agents targeting the large protein interaction interfaces engaged by the fungal GTPase.

Amino Acid Sequence↗

PubMed2005

Solution structure of the ligand binding domain of the fibroblast growth factor receptor: role of heparin in the activation of the receptor.

The three-dimensional solution structure of the ligand binding D2 domain of the fibroblast growth factor receptor (FGFR) is determined using multidimensional NMR techniques. The atomic root-mean-square distribution for the backbone atoms in the structured region is 0.64 A. Secondary structural elements in the D2 domain include 11 beta-strands arranged antiparallely into two layers of beta-sheets. The structure of the D2 domain is characterized by the presence of a short flexible helix that protrudes out of the layers of beta-sheets. Results of size exclusion chromatography and sedimentation velocity experiments show that the D2 domain exists in a monomeric state both in the presence and in the absence of bound sucrose octasulfate (SOS), a structural analogue of heparin. Comparison of the solution structure of the D2 domain with the crystal structure of the protein (D2 domain) in the FGF signaling complex reveals significant differences, suggesting that ligand (FGF) binding may induce significant conformational changes in the receptor. SOS binding sites in the D2 domain have been mapped on the basis of the 1H-15N chemical shift perturbation data. SOS binds to the positively charged residues located in beta-strand III and the flexible helix. Isothermal titration calorimetry data indicate that the ligand (hFGF-1) binds strongly (Kd approximately 10(-9) M) to the D2 domain even in the absence of SOS. Binding of SOS to either the D2 domain or hFGF-1 does not seem to be the driving force for the formation of the D2-hFGF-1 binary complex. The function of SOS binding appears to stabilize the preformed D2-FGF binary complex.

Binding Sites↗

PubMed2005

Isolation of monomeric human V(H)s by a phage selection.

Human V(H) domains are promising molecules in applications involving antibodies, in particular, immunotherapy because of their human origin. However, they are, in general, prone to aggregation. Therefore, various strategies have been employed to acquire monomeric human V(H)s. We had previously discovered that filamentous phages displaying engineered monomeric V(H) domains gave rise to significantly larger plaques on bacterial lawns than phages displaying wild type V(H)s with aggregation tendencies. Using plaque size as the selection criterion and a phage-displayed naïve human V(H) library we identified 15 V(H)s that were monomeric. Additionally, the V(H)s demonstrated good expression yields, good refolding properties following thermal denaturation, resistance to aggregation during long incubation at 37 degrees C, and to trypsin at 37 degrees C. These 15 V(H)s should serve as good scaffolds for developing immunotherapeutics, and the selection method employed here should have general utility for isolating proteins with desirable biophysical properties.

Amino Acid Sequence↗

PubMed2005

Transforming bivalent ligands into retractable enzyme inhibitors through polypeptide-protein interactions.

The concept of bivalent polypeptides with controllable flexible linkers is demonstrated through the design of a new generation of 'antidote'-reversible inhibitors of thrombin. These molecules contain two binding moieties, each of which in isolation has only a moderate affinity of binding, which are linked together by a flexible peptide bridge. We show that activities of the potent bivalent inhibitors of thrombin can be reversed by the specific, but much weaker, binding of the linker moiety to protein 'antidotes'.

Amino Acid Sequence↗

PubMed2005

Polymerization of the SAM domain of MAPKKK Ste11 from the budding yeast: implications for efficient signaling through the MAPK cascades.

The sterile alpha-motif (SAM) is a protein module approximately 70 residues long and mainly involved in the protein-protein interactions of cell signaling and transcriptional repression. The SAM domain of the yeast MAPKKK Ste11 has a well-folded dimeric structure in solution. Interestingly, the well-folded dimer of the Ste11 SAM undergoes a time-dependent self-assembly upon lowering of the pH, leading to the formation of high molecular weight oligomers. The oligomeric structures rapidly disassemble to the well-folded dimer upon reversal of the pH to close to neutral conditions. Circular dichroism (CD) and atomic force microscopy (AFM) experiments demonstrate that the oligomeric structure formed at pH 5.0 appears to be highly helical and has architecture akin to proto-fibrils. Residue-specific kinetics of pH-triggered oligomerization obtained from real-time 15N-1H HSQC experiments indicate that the dimer-oligomer transition appears to involve all residues of the well-folded dimeric structure of the Ste11 SAM. Very interestingly, the interactions of the Ste11 and Ste50 SAM domains also lead to the formation of non-homogeneous hetero-complexes with significant populations of high molecular weight aggregates. AFM imaging shows that the Ste11-Ste50 hetero-polymeric aggregates assume the shapes of circular nano-particles with dimensions of 50-60 nano-meters (nm), in contrast to the proto-fibrils formed by the Ste11 SAM domain alone. Such intrinsic propensity for dimer to oligomer transition of the Ste50-binding SAM domain of Ste11 may endow the MAPKKK Ste11 with unique functional properties required for efficient and high fidelity signal transduction in the budding yeast.

Circular Dichroism↗

PubMed2005

Analysis of the phosphoryl transfer mechanism of c-AMP dependent protein kinase (PKA) by penta-coodinate phosphoric transition state theory.

This review briefly covers recent literature of research on the phosphoryl transfer mechanism of PKA. Combining experimental and theoretical calculation results on enzymes with experimentally observed biomimic activities of phosphoryl amino acids and a small molecular model of catalytic core in PKA, a novel mechanism was proposed. The cooperative participation roles of both Asp166 and Lys168 via a penta-coodinate phosphoric intermediate was elucidated to conciliate the current different views of the phosphoryl transfer mechanism of PKA. Since many ATP-binding enzymes may share a similar phosphoryl transfer mechanism, this proposed mechanism might also apply to the mechanism of these enzymes, e.g., molecular motor and phosphatase among others.

Catalysis↗

PubMed2004

Solution structure of the dimeric SAM domain of MAPKKK Ste11 and its interactions with the adaptor protein Ste50 from the budding yeast: implications for Ste11 activation and signal transmission through the Ste50-Ste11 complex.

Ste11, a homologue of mammalian MAPKKKs, together with its binding partner Ste50 works in a number of MAPK signaling pathways of Saccharomyces cerevisiae. Ste11/Ste50 binding is mediated by their sterile alpha motifs or SAM domains, of which homologues are also found in many other intracellular signaling and regulatory proteins. Here, we present the solution structure of the SAM domain or residues D37-R104 of Ste11 and its interactions with the cognate SAM domain-containing region of Ste50, residues M27-Q131. NMR pulse-field-gradient (PFG) and rotational correlation time measurements (tauc) establish that the Ste11 SAM domain exists predominantly as a symmetric dimer in solution. The solution structure of the dimeric Ste11 SAM domain consists of five well-defined helices per monomer packed into a compact globular structure. The dimeric structure of the SAM domain is maintained by a novel dimer interface involving interactions between a number of hydrophobic residues situated on helix 4 and at the beginning of the C-terminal long helix (helix 5). The dimer structure may also be stabilized by potential salt bridge interactions across the interface. NMR H/2H exchange experiments showed that binding of the Ste50 SAM to the Ste11 SAM very likely involves the positively charged extreme C-terminal region as well as exposed hydrophobic patches of the dimeric Ste11 SAM domain. The dimeric structure of the Ste11 SAM and its interactions with the Ste50 SAM may have important roles in the regulation and activation of the Ste11 kinase and signal transmission and amplifications through the Ste50-Ste11 complex.

Amino Acid Sequence↗

PubMed2004

HCV NS5B polymerase-bound conformation of a soluble sulfonamide inhibitor by 2D transferred NOESY.

HCV NS5B RNA-dependent RNA polymerase (NS5B) is essential for viral replication and is therefore considered a target for antiviral drug development. From our ongoing screening effort in the search for new anti-HCV agents, a novel inhibitor 1 with low microM activity against the HCV NS5B polymerase was identified. SAR analysis indicated the optimal substitution pattern required for activity, for example, carboxylic acid group at 2-position of thiophene ring. We describe the steps taken to identify and solve the bioactive conformation of derivative 6 through the use of the transferred NOE method (trNOE).

Antiviral Agents↗

PubMed2004

Rational design and selection of bivalent peptide ligands of thrombin incorporating P4-P1 tetrapeptide sequences: from good substrates to potent inhibitors.

The tetrapeptide Phe-Asn-Pro-Arg is a structurally optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or some variants to a C-terminal fragment of hirudin, we were able to generate a series of new bivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P3 and P3' sites of the active-site directed sequence, Phe(P4)-Xaa(P3)-Pro(P2)-Arg(P1)-Pro(P1')-Gln(P2')-Yaa(P3'). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P4, P3 and P3' sites for bivalent and optimized two-site binding. Very significantly, panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-clotting activities in the low nanomolar range and yet interfering only partially the catalytic active site of thrombin. Modes of action of the newly discovered bivalent inhibitors are rationalized in light of the allosteric properties of thrombin, especially the interplay between the proteolytic action and regulatory binding occurring at thrombin surfaces remote from the catalytic active site.

Amino Acid Sequence↗

PubMed2004

Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein.

The B class cell-attached ephrins mediate contact-dependent cell-cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified: one contains the PSD-95/Dlg/ZO-1 (PDZ) domain (for example PDZ-RGS3), and the second the Src homology 2 (SH2) domain (e.g. the Grb4 adaptor protein). The interaction with Grb4 requires phosphorylation of tyrosine residues on the conserved cytoplasmic C-terminal region of B ephrins, while binding to the PDZ domain is independent of tyrosine phosphorylation. However, the exact phosphorylation site(s) required for signaling remained obscure and it is also unknown whether the two classes of effectors can bind to B ephrins simultaneously or if the binding of one affects the binding of the other. We report here that phosphorylation of Tyr304 in the functional C-terminal region (residues 301-333) of ephrin B2 confers high-affinity binding to the SH2 domain of the Grb4 protein. Tyrosine phosphorylation at other candidate sites resulted in only minor change of the binding of Tyr304-phosphorylated ephrin B peptide (i.e. ephrinB2(301-333)-pY304) with the SH2 domain. (1)H-(15)N NMR HSQC experiments show that only the ephrinB2(301-333)-pY304 peptide forms a stable and specific binding complex with the SH2 domain of Grb4. The SH2 and PDZ domains were found to bind to the Tyr304 phosphopeptide both independently and at the same time, forming a three-component molecular complex. Taken together, our studies identify a novel SH2 domain binding motif, PHpY304EKV, on the cytoplasmic domains of B ephrins that may be essential for reverse signaling via the Grb4 adaptor protein alone or in concert with proteins containing PDZ domains.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Probing the kinetic landscape of transient peptide-protein interactions by use of peptide (15)n NMR relaxation dispersion spectroscopy: binding of an antithrombin peptide to human prothrombin.

Protein-ligand interactions may lead to the formation of multiple molecular complexes in dynamic exchange, affecting the kinetic and thermodynamic characteristics of the binding equilibrium. We followed the dissociation kinetics of the transient and specific complex of an antithrombotic peptide N-acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro(60)-Glu-Glu-Tyr-Leu-Gln(65) with human prothrombin by use of (15)N NMR relaxation dispersion spectroscopy of the peptide. Every one of the five (15)N-labeled adjacent residues of the peptide exhibited apparently different kinetic exchange and relaxation behaviors, which were especially evident at different concentrations of prothrombin. Binding-induced (15)N relaxation dispersion of residues Phe(56), Glu(57), Glu(58), and Ile(59) can be fitted phenomenologically to a two-site on-and-off exchange mechanism with physically feasible relaxation and kinetic parameters obtained for residues Phe(56), Glu(58), and Ile(59), independent of the prothrombin concentration. The apparent kinetic parameters of Glu(57) show some dependence on the concentration of prothrombin and the extracted transverse relaxation rate for Glu(57) in the bound state was severalfold higher than that expected for a protein-peptide complex with a size of approximately 72 kDa. In addition, the equilibrium population of the bound peptide obtained for Glu(57) was inconsistent with those for Phe(56), Glu(58), and Ile(59) and with the prothrombin/peptide ratios used in the experiments. These discrepancies can be explained by the presence of two conformations for the peptide-protein complex exchanging at a rate of approximately 100 s(-)(1). In all, our study shows that fast dissociation of protein-peptide complexes can be studied quantitatively using peptide (15)N NMR relaxation dispersion measurements without a precise knowledge of the peptide and protein concentrations. In addition, protein titration was found to improve the accuracy of quantitative analysis and may make it possible to determine the rate of conformational changes within the protein-peptide complex.

Antithrombins↗

PubMed2003

Unexpected binding mode for 2'-phosphoadenosine-based nucleotide inhibitors in complex with human angiogenin revealed by heteronuclear NMR spectroscopy.

Human angiogenin (Ang) is a tumor-promoting RNase in the pancreatic RNase superfamily. Efforts to develop nucleotide-based inhibitors of Ang as potential anticancer drugs have been hampered by the lack of direct structural information on Ang-nucleotide complexes. Here, we have used heteronuclear NMR spectroscopy with (15)N- and (15)N/(13)C-labeled Ang to map the interactions of Ang with the phosphate ion, seven adenosine mononucleotides (the 2'-, 3'-, and 5'-monophosphates, the 2',5'- and 3',5'-diphosphates, the 5'-diphosphate, and the 2'-monophospho-5'-diphosphate), and the dinucleotide 2'-deoxyuridine 3'-pyrophosphate (P' --> 5') adenosine-2'-phosphate (dUppA-2'-p). The 2'-phosphate based derivatives, which bind more tightly than the corresponding 3'-phosphate isomers, induced characteristic large resonance perturbations of the backbone amide proton of Leu(115), the backbone (15)N of His(114), and the Gln(12) side-chain NH(2) group in the Ang active site. In contrast, adenosine derivatives with only 3'- or 5'-phosphates produced much less dramatic perturbations of Leu(115) and His(114) resonances, along with modest perturbations of additional residues both within and beyond the active site. Measurements of NOEs together with molecular docking analyses revealed the three-dimensional structures of the complexes of Ang with adenosine 2',5'-diphosphate and dUppA-2'-p; the binding modes of these inhibitors differ substantially from those predicted in earlier studies. Most notably, the 2'-phosphate rather than the 5'-phosphate occupies the P(1) catalytic subsite of Ang, and the side chain of His(114) has undergone a conformational transition that positions it outside P(1) and allows it to form stacking interactions with the adenine ring of the inhibitor. Strikingly, the 2'-deoxyuridine moiety of dUppA-2'-p makes only a few contacts with Ang, and these involve residues outside the B(1) subsite where the pyrimidine ring of substrates normally binds.

Adenine Nucleotides↗

PubMed2003

Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans.

The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30-40 mg/l in LB, 15-20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with (15)N, (15)N/(13)C and even (15)N/(13)C/(2)H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.

Amino Acid Sequence↗

PubMed2002

Stability of protein-bound conformations of bioactive peptides: the folded conformation of an epidermal growth factor-like thrombomodulin fragment is similar to that recognized by thrombin.

The relationship between the free and bound conformations of bioactive peptides is explored using the epidermal growth factor (EGF)-like thrombomodulin fragment hTM409-426 as a model system. The hTM409-426 peptide has a sequence of C(409)PEGYILDDGFIC(421)TDIDE (with a disulfide bond between Cys409 and Cys421) and is a selective inhibitor of thrombin. Upon binding to thrombin, hTM409-426 adopts a well-defined conformation-namely, a beta-turn followed by an antiparallel beta-sheet, similar to those found in all other EGF-like protein repeats (Hrabal et al., Protein Science, 1996, Vol. 5, 195-203). Here we demonstrate that, at pH 6.8 and at 25 degrees C, the hTM409-426 peptide in the free state is very flexible, but still populates a type II beta-turn over residues Pro410-Glu411-Gly412-Tyr413 and the clustering of some hydrophobic side chains, both of which are present in the thrombin-bound conformation. At a lower temperature of 5 degrees C, significant conformational shifts of the C alpha H proton resonances and extensive medium- and long-range NOEs are observed, indicating the presence of folded conformations with unique backbone-backbone and side-chain interactions. A comparison of the NOE patterns in the free state with transferred NOEs shows that the free-state folded and the thrombin-bound conformations of the hTM409-426 peptide are very similar, particularly over residues Pro410-Ile424. The folded conformation of hTM409-426 appears to be stabilized by two hydrophobic clusters, one formed by the side chains of residues Pro410, Tyr413, Leu415, and Phe419 and the Cys409-Cys421 disulfide bond, the second involving residues Ile414 and Ile424. These results indicate that the overall topology of the thrombin-bound conformation of the hTM409-426 peptide is prefolded in the free state and the primary sequence (including the disulfide bond) may be selective for an ensemble of conformations similar to that recognized by thrombin.

Magnetic Resonance Spectroscopy↗

PubMed2002

Solution structure and backbone dynamics of the functional cytoplasmic subdomain of human ephrin B2, a cell-surface ligand with bidirectional signaling properties.

The cytoplasmic domain of B ephrins plays a central role in bidirectional signal transduction processes controlling pattern formation and morphogenesis, such as axon guidance, cell migration, segmentation, and angiogensis. In particular, the extremely conserved last 33-residue cytoplasmic subdomain was shown to bind to both a PDZ domain for one signaling pathway [Lu et al. (2001) Cell 105, 69-79] and an SH2 domain from an alternative signaling network [Cowan and Henkemeyer (2001) Nature 413, 174-179]. To date, no structural information is available for the cytoplasmic domain of ephrin B proteins. We report here a detailed NMR study on the structural and dynamic properties of the cytoplasmic domain of human ephrin B2. Our results reveal the following: (1) the N-terminal region of the cytoplasmic domain from residues 253 to 300 lacks the ability for structure formation and is particularly prone to aggregation; and (2) the C-terminal functional subdomain from residues 301 to 333 assumes two distinctive structural elements with residues 301-322 adopting a well-packed hairpin structure followed by a flexible C-terminal tail. Furthermore, the backbone (15)N relaxation data demonstrate that the hairpin structure has significantly limited backbone motions, indicating a high conformational stability for the folded structure. Therefore, while the flexible C-terminal tail is suitable for binding to the PDZ domain, the folded hairpin may represent a latent structure requiring phosphorylation-induced conformational changes for high-affinity interactions with the SH2 domain.

Amino Acid Sequence↗