Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV-1 capsid protein.

The cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV-1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His-Ala-Gly-Pro-Ile-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X-ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV-1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.     

Functions of the HIV-1 matrix protein p17

HIV-1 replication is a dynamic process influenced by a combination of viral and host factors. The HIV-1 matrix protein p17 is a structural protein critically involved in most stages of the life cycle of the retrovirus. It participates in the early stages of virus replication as well as in RNA targeting to the plasma membrane, incorporation of the envelope into virions and particle assembly. Besides its well established functions, p17 acts as a viral cytokine that works on preactivated--but not on resting--human T cells promoting proliferation, proinflammatory cytokines release and HIV-1 replication after binding to a cellular receptor (p17R). Thus, p17 might play a key role in the complex network of host- and virus-derived stimulatory factors contributing to create a favourable environment for HIV-1 infection and replication. Here, we present a brief overview of the functions played by the matrix protein p17 in the HIV-1 life cycle and summarize the current understanding of how p17 could contribute to the pathogenesis of HIV-1 disease.     

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

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

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

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

HIV-1 assembly initiated by p17 matrix protein

HIV-1 matrix protein (MA) is a multifunctional structural protein located on the N-terminus of Gag precursor p55 and is responsible for its transport to the plasma membrane, the site of virus assembly. In the present paper, it has been shown that MA is cleaved from Gag precursor at an early stage of the virus infection and participates in virus assembly. MA is transported into the nuclei wherein it associates with viral RNA (vRNA). The MA-vRNA complex is transported to the plasma membrane. Mutant MA, which lost its membranotropic signal, does not reach the plasma membrane and MA-vRNA complex remains in the nuclei and cytoskeleton. Thus, MA seems to deliver vRNA from the nuclei to plasma membrane through the cytoskeleton, initiating virus assembly.     

Solution structure of Rap1 BRCT domain from Saccharomyces cerevisiae reveals a novel fold

Rap1 (repressor-activator protein 1) from Saccharomyces cerevisiae, containing a BRCT domain at its N-terminus, is a multifunctional protein that controls telomere function, silencing, and the activation of glycolytic and ribosomal protein genes. In this work, we determined the solution structure of Rap1 BRCT domain, which contains three β-strands and three α-helices. Structural comparison indicated that Rap1 BRCT domain adopts a global fold similar to other BRCT domains, implying some common structural aspects of BRCT domain family. On the other hand, Rap1 BRCT domain displays structural characteristics significantly different from other BRCT domains in that Rap1 BRCT domain adopts a rather flexible conformation with less secondary structure elements, revealing a novel fold of the BRCT domain family.     

Small-angle X-ray scattering reveals the N-terminal domain organization of cardiac myosin binding protein C

Myosin binding protein C (MyBP-C) is a multidomain accessory protein of striated muscle sarcomeres. Three domains at the N-terminus of MyBP-C (C1-m-C2) play a crucial role in maintaining and modulating actomyosin interactions. The cardiac isoform has an additional N-terminal domain (C0) that is postulated to provide a greater level of regulatory control in cardiac muscle. We have used small-angle X-ray scattering, ab initio shape restoration, and rigid-body modeling to determine the average shape and spatial arrangement of the four N-terminal domains of cardiac MyBP-C (C0C2) and a three-domain variant that is analogous to the N-terminus of the skeletal isoform (C1C2). We found that the domains of both proteins are tandemly arranged in a highly extended configuration that is sufficiently long to span the interfilament cross-bridge distances in vivo and, hence, be poised to modulate these interactions. The average spatial organization of the C1, m, and C2 domains is not significantly perturbed by the removal of the cardiac-specific C0 domain, suggesting that the interdomain interfaces, while relatively small in area, have a degree of rigidity. Modeling the C0C2 and C1C2 scattering data reveals that the structures of the C0 and m domains (also referred to as the ‘MyBP motif’) are compact and have dimensions that are consistent with the immunoglobulin fold superfamily of proteins. Sequence analysis, homology modeling, and circular dichroism experiments support the conclusion that the previously undetermined structures of these domains can be characterized as having an immunoglobulin-like fold. Atomic models using the known NMR structures for C1 and C2 as well as homology models for the C0 and m domains provide insights into the placement of conserved serine residues of the m domain that are phosphorylated in vivo and cause a change in muscle fiber contraction by abolishing interactions with myosin.     

X-ray structure of a novel matrix metalloproteinase inhibitor complexed to stromelysin

A new class of matrix metalloproteinase (MMP) inhibitors has been identified by screening a collection of compounds against stromelysin. The inhibitors, 2,4,6-pyrimidine triones, have proven to be potent inhibitors of gelatinases A and B. An X-ray crystal structure of one representative compound bound to the catalytic domain of stromelysin shows that the compounds bind at the active site and ligand the active-site zinc. The pyrimidine triones mimic substrates in forming hydrogen bonds to key residues in the active site, and provide opportunities for placing appropriately chosen groups into the S1' specificity pocket of MMPS: A number of compounds have been synthesized and assayed against stromelysin, and the variations in potency are explained in terms of the binding mode revealed in the X-ray crystal structure.     

HIV-1 assembly is initiated by p17 matrix protein

HIV-1 matrix protein (MA) is multifunctional structural protein located on N-terminus of Gag precursor p55 and responsible for its transport to plasma membrane, the site of virus assembly. Here, it has been shown that MA is cleaved from Gag precursor at early stage of the virus infection and participates in virus assembly. MA is transported into the nuclei wherein it associates with viral RNA (vRNA). The MA-vRNA complex is transported to plasma membrane. Mutant MA which lost its membranotropic signal does not reach plasma membrane and MA-vRNA complex remains in the nuclei and cytoskeleton. Thus, MA seems to deliver vRNA from the nuclei to plasma membrane through cytoskeleton initiating virus assembly.     

Solution structure of the HIV-1 integrase-binding domain in LEDGF/p75

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase (IN) in human cells. We have determined the NMR structure of the integrase-binding domain (IBD) in LEDGF and identified amino acid residues essential for the interaction. The IBD is a compact right-handed bundle composed of five alpha-helices. Based on folding topology, the IBD is structurally related to a diverse family of alpha-helical proteins that includes eukaryotic translation initiation factor eIF4G and karyopherin-beta. LEDGF residues essential for the interaction with IN were localized to interhelical loop regions of the bundle structure. Interaction-defective IN mutants were previously shown to cripple replication although they retained catalytic function. The initial structure determination of a host cell factor that tightly binds to a retroviral enzyme lays the groundwork for understanding enzyme-host interactions important for viral replication.     

Solution structure of the PDZ2 domain from cytosolic human phosphatase hPTP1E complexed with a peptide reveals contribution of the beta2-beta3 loop to PDZ domain-ligand interactions

The solution structure of the second PDZ domain from human phosphatase hPTP1E in complex with a C-terminal peptide from the guanine nucleotide exchange factor RA-GEF-2 has been determined using 2D and 3D heteronuclear NMR experiments. Compared to previously solved structures, the hPTP1E complex shows an enlarged interaction surface with the C terminus of the bound peptide. Novel contacts were found between the long structured beta2/beta3 loop of the PDZ domain and the sixth amino acid residue from the C terminus of the peptide. This work underlines the importance of the beta2/beta3 loop for ligand selection by PDZ domains.     

Crystal structure of d(GGCCAATTGG) complexed with DAPI reveals novel binding mode

The single-crystal X-ray structure of the complex between the minor groove binder 4',6-diamidino-2-phenylindole (DAPI) and d(GGCCAATTGG) reveals a novel way of off-centered binding, with an unique hydrogen bond between the minor groove binder and a CG base pair. Application of crystal engineering and cryocooling techniques helped to extend the resolution to 1.9 A, resulting in an unambiguous determination of drug conformation and orientation. The structure was refined to completion using SHELXL-93, resulting in a residual factor R of 18. 0% for 3562 reflections with F(o) > 4sigma(F(o)) including 81 water molecules. As the bulky NH(2)-group on guanine is believed to prevent drug binding in the minor groove, the nature and stability of the CG-DAPI contact was further addressed in full detail using ab initio quantum chemical methods. The amino groups involved in the guanine-drug interaction are substantially nonplanar, resulting in an energy gain of about 5 kcal/mol. The combined structural and theoretical data suggest that the guanine NH(2)-group does not destabilize the drug binding to an extent that it prevents complexation.     

X-ray structure of HIV-1 protease tethered dimer complexed to ritonavir

We report here the 2.5A structure of HIV-1 protease tethered-dimer ritonavir complex. The inhibitor bound in the active site has different conformations in the two orientations. There is only one hydrogen bond between the inhibitor and the enzyme. The conserved flap-water is not found in the present complex.     

Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid

The human immunodeficiency virus type 1 (HIV-1) capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study, we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1, for the capsid protein (K(d) approximately 1 mum) and demonstrated its ability to penetrate the cell membrane (Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., Heck, S., Goger, M., Cowburn, D., Freed, E. O., and Debnath, A. K. (2008) J. Mol. Biol. 378, 565-580). In cell-based assays, NYAD-1 colocalized with the Gag polyprotein during traffic to the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent-exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly.     

Solution structure of Kti11p from Saccharomyces cerevisiae reveals a novel zinc-binding module

Kti11p is a small, highly conserved CSL zinc finger-containing protein found in many eukaryotes. It was first identified as one of the factors required for maintaining the sensitivity of Saccharomyces cerevisiae to Kluyveromyces lactis zymocin. Then, it was found to be identical to Dph3, a protein required for diphthamide biosynthesis on eEF-2, the target of diphtheria toxin and Pseudomonas exotoxin A, in both yeast and higher eukaryotes. Furthermore, Kti11p/Dph3 was found to physically interact with core-Elongator, ribosomal proteins, eEF-2, two other proteins required for diphthamide modification on eEF-2, and DelGEF. Here, we determined the solution structure of Kti11p using NMR, providing the first structure of the CSL-class zinc-binding protein family. We present the first experimental evidence that Kti11p can bind a single Zn(2+) ion by its four conserved cysteine residues. The major structure of Kti11p comprises a beta sandwich as well as an alpha helix. Moreover, a structure-based similarity search suggests that it represents a novel structure and may define a new family of the zinc ribbon fold group. Therefore, our work provides a molecular basis for further understanding the multiple functions of Kti11p/Dph3 in different biological processes.     

Solution structure of the SH2 domain of Grb2 complexed with the Shc-derived phosphotyrosine-containing peptide.

The solution structure of growth factor receptor-bound protein 2 (Grb2) SH2 complexed with a Shc-derived phosphotyrosine (pTyr)-containing peptide was determined by nuclear magnetic resonance (NMR) spectroscopy. The pTyr binding site of Grb2 SH2 was similar to those of other SH2 domains. In contrast, the amino acid residues C-terminal to pTyr did not form an extended structure because of steric hindrance caused by a bulky side-chain of Trp121 (EF1). As a result, the peptide formed a turn-structure on the surface of Grb2 SH2. The asparagine residue at the pTyr+2 position of the Shc-peptide interacted with the main-chain carbonyl groups of Lys109 and Leu120. The present solution structure was similar to the crystal structure reported for Grb2 SH2 complexed with a BCR-Abl-derived phosphotyrosine-containing peptide. Finally, the structure of Grb2 SH2 domain was compared with those of the complexes of Src and phospholipase C-gamma1 with their cognate peptides, showing that the specific conformation of the peptide was required for binding to the SH2 domains.     

Solution structure and backbone dynamics of the catalytic domain of matrix metalloproteinase-2 complexed with a hydroxamic acid inhibitor

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.     

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

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

Solution structure of a double mutant of the carboxy-terminal dimerization domain of the HIV-1 capsid protein

As in other retroviruses, the HIV-1 capsid (CA) protein is composed of two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), joined by a flexible linker. The dimerization of the CTD is thought to be a critical step in the assembly of the immature and mature viral capsids. The precise nature of the functional form of CTD dimerization interface has been a subject of considerable interest. Previously, the CTD dimer was thought to involve a face-to-face dimerization observed in the early crystallographic studies. Recently, the crystallographic structure for a domain-swapped CTD dimer has been determined. This dimer, with an entirely different interface that includes the major homology region (MHR) has been suggested as the functional form during the Gag assembly. The structure determination of the monomeric wt CTD of HIV-1 has not been possible because of the monomer-dimer equilibrium in solution. We report the NMR structure of the [W184A/M185A]-CTD mutant in its monomeric form. These mutations interfere with dimerization without abrogating the assembly activity of Gag and CA. The NMR structure shows some important differences compared to the CTD structure in the face-to-face dimer. Notably, the helix-2 is much shorter, and the kink seen in the crystal structure of the wt CTD in the face-to-face dimer is absent. These NMR studies suggest that dimerization-induced conformational changes may be present in the two crystal structures of the CTD dimers and also suggest a mechanism that can simultaneously accommodate both of the distinctly different dimer models playing functional roles during the Gag assembly of the immature capsids.     

Binding of calmodulin to the HIV-1 matrix protein triggers myristate exposure

Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca(2+)-binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(+)MA) domain. Herein, we demonstrate that CaM binds to myr(+)MA with a dissociation constant (K(d)) of ～2 μm and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(+)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.