CCR2 and CCR5 receptor-binding properties of herpesvirus-8 vMIP-II based on sequence analysis and its solution structure.

Human herpesvirus-8 (HHV-8) is the infectious agent responsible for Kaposi's sarcoma and encodes a protein, macrophage inflammatory protein-II (vMIP-II), which shows sequence similarity to the human CC chemokines. vMIP-II has broad receptor specificity that crosses chemokine receptor subfamilies, and inhibits HIV-1 viral entry mediated by numerous chemokine receptors. In this study, the solution structure of chemically synthesized vMIP-II was determined by nuclear magnetic resonance. The protein is a monomer and possesses the chemokine fold consisting of a flexible N-terminus, three antiparallel beta strands, and a C-terminal alpha helix. Except for the N-terminal residues (residues 1-13) and the last two C-terminal residues (residues 73-74), the structure of vMIP-II is well-defined, exhibiting average rmsd of 0.35 and 0.90 A for the backbone heavy atoms and all heavy atoms of residues 14-72, respectively. Taking into account the sequence differences between the various CC chemokines and comparing their three-dimensional structures allows us to implicate residues that influence the quaternary structure and receptor binding and activation of these proteins in solution. The analysis of the sequence and three-dimensional structure of vMIP-II indicates the presence of epitopes involved in binding two receptors CCR2 and CCR5. We propose that vMIP-II was initially specific for CCR5 and acquired receptor-binding properties to CCR2 and other chemokine receptors.     

A novel peptide antagonist of CXCR4 derived from the N-terminus of viral chemokine vMIP-II

The viral macrophage inflammatory protein-II (vMIP-II) encoded by Kaposi's sarcoma-associated herpesvirus is unique among all known chemokines in that vMIP-II shows a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus type 1 (HIV-1). To elucidate the mechanism of the promiscuous receptor interaction of vMIP-II, synthetic peptides derived from the N-terminus of vMIP-II were studied. In contrast to the full-length protein that recognizes both CXCR4 and CCR5, a peptide corresponding to residues 1-21 of vMIP-II (LGASWHRPDKCCLGYQKRPLP) was shown to strongly bind CXCR4, but not CCR5. The IC(50) of this peptide in competing with CXCR4 binding of (125)I-SDF-1alpha is 190 nM as compared to the IC(50) of 14.8 nM of native vMIP-II in the same assay. The peptide selectively prevented CXCR4 signal transduction and coreceptor function in mediating the entry of T- and dual-tropic HIV-1 isolates, but not those of CCR5. Further analysis of truncated peptide analogues revealed the importance of the first five residues for the activity with CXCR4. These results suggest that the N-terminus of vMIP-II is essential for its function via CXCR4. In addition, they reveal a possible mechanism for the distinctive interactions of vMIP-II with different chemokine receptors, a notion that may be further exploited to dissect the structural basis of its promiscuous biological function. Finally, the potent CXCR4 peptide antagonist shown here could serve as a lead for the development of new therapeutic agents for HIV infection and other immune system diseases.     

Structural and functional characterization of human CXCR4 as a chemokine receptor and HIV-1 co-receptor by mutagenesis and molecular modeling studies

The human CXC chemokine receptor 4 (CXCR4) is a receptor for the chemokine stromal cell-derived factor (SDF-1alpha) and a co-receptor for the entry of specific strains of human immunodeficiency virus type I (HIV-1). CXCR4 is also recognized by an antagonistic chemokine, the viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus type VIII. SDF-1alpha or vMIP-II binding to CXCR4 can inhibit HIV-1 entry via this co-receptor. An approach combining protein structural modeling and site-directed mutagenesis was used to probe the structure-function relationship of CXCR4, and interactions with its ligands SDF-1alpha and vMIP-II and HIV-1 envelope protein gp120. Hypothetical three-dimensional structures were proposed by molecular modeling studies of the CXCR4.SDF-1alpha complex, which rationalize extensive biological information on the role of CXCR4 in its interactions with HIV-1 envelope protein gp120. With site-directed mutagenesis, we have identified that the amino acid residues Asp (D20A) and Tyr (Y21A) in the N-terminal domain and the residue Glu (E268A) in extracellular loop 3 (ECL3) are involved in ligand binding, whereas the mutation Y190A in extracellular loop 2 (ECL2) impairs the signaling mediated by SDF-1alpha. As an HIV-1 co-receptor, we found that the N-terminal domain, ECL2, and ECL3 of CXCR4 are involved in HIV-1 entry. These structural and mutational studies provide valuable information regarding the structural basis for CXCR4 activity in chemokine binding and HIV-1 viral entry, and could guide the design of novel targeted inhibitors.     

Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.

Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an antagonist of CCR5 coreceptor activity. These findings suggest that the molecular anatomy of CCR5 binding plays a critical role in antagonism of coreceptor activity.     

NMR structures of anti-HIV D-peptides derived from the N-terminus of viral chemokine vMIP-II

The viral macrophage inflammatory protein-II (vMIP-II) encoded by Kaposi's sarcoma-associated herpesvirus has unique biological activities in that it blocks the cell entry by several different human immunodeficiency virus type 1 (HIV-1) strains via chemokine receptors including CXCR4 and CCR5. In this paper, we report the solution structure of all-d-amino acid peptides derived from the N-terminus of vMIP-II, which have been shown to have strong CXCR4 binding activity and potently inhibit HIV-1 entry via CXCR4, by using long mixing time two-dimensional nuclear Overhauser enhancement spectroscopy experiments. Both of all-d-peptides vMIP-II (1-10) and vMIP-II (1-21), which are designated as DV3 and DV1, respectively, have higher CXCR4 binding ability than their l-peptide counterparts. They are partially structured in aqueous solution, displaying a turn-like structure over residues 5-8. The small temperature coefficients of His-6 amide proton for both peptides also suggest the formation of a small hydrophobic pocket centered on His-6. The structural features of DV3 are very similar to the reported solution structure of all-l-peptide vMIP-II (1-10) [M.P. Crump, E. Elisseeva, J. Gong, I. Clark-Lewis, B.D. Sykes, Structure/function of human herpesvirus-8 MIP-II (1-71) and the antagonist N-terminal segment (1-10), FEBS Lett. 489 (2001) 171], which is consistent with the notion that d- and l-enantiomeric peptides can adopt mirror image conformations. The NMR structures of the d-peptides provide a structural basis to understand their mechanism of action and design new peptidomimetic analogs to further explore the structure-activity relationship of d-peptide ligand binding to CXCR4.     

Structure/function of human herpesvirus-8 MIP-II (1-71) and the antagonist N-terminal segment (1-10)

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II that has been shown to be a broad range human chemokine receptor antagonist. Two N-terminal peptides, vMIP-II(1-10) and vMIP-II(1-11)dimer (dimerised through Cys11) were synthesised. Both peptides are shown to bind the CXC chemokine receptor 4 (CXCR4). vMIP-II(1-10) was 1400-fold less potent than the native protein whilst the vMIP-II(1-11)dimer was only 180-fold less potent. In addition, both peptides are CXCR4 antagonists. Through analysis of non-standard, long mixing time two-dimensional nuclear Overhauser enhancement spectroscopy experiments, 13C relaxation data and amide chemical shift temperature gradients for the N-terminus of vMIP-II, we show that this region populates a turn-like structure over residues 5-8, both in the presence and absence of the full protein scaffold. This major conformation is likely to be in fast exchange with other conformational states but it has not previously been detected in monomeric chemokine structures. This and other studies [Elisseeva et al. (2000) J. Biol. Chem. 275, 26799-26805] suggest that there may be a link between the structuring of the short N-terminal chemokine peptides and their ability to bind their receptor.     

Helical interactions in the HIV-1 gp41 core reveal structural basis for the inhibitory activity of gp41 peptides

The HIV-1 gp41 envelope protein mediates membrane fusion that leads to virus entry into the cell. The core structure of fusion-active gp41 is a six-helix bundle in which an N-terminal three-stranded coiled coil is surrounded by a sheath of antiparallel C-terminal helices. A conserved glutamine (Gln 652) buried in this helical interface replaced by leucine increases HIV-1 infectivity. To define the basis for this enhanced membrane fusion activity, we investigate the role of the Gln 652 to Leu substitution on the conformation, stability, and biological activity of the N34(L6)C28 model of the gp41 ectodomain core. The 2.0 A resolution crystal structure of the mutant molecule shows that the Leu 652 side chains make prominent contacts with hydrophobic grooves on the surface of the central coiled coil. The Gln 652 to Leu mutation leads to a marginal stabilization of the six-helix bundle by -0.8 kcal/mol, evaluated from thermal unfolding experiments. Strikingly, the mutant N34(L6)C28 peptide is a potent inhibitor of HIV-1 infection, with 10-fold greater activity than the wild-type molecule. This inhibitory potency can be traced to the corresponding C-terminal mutant peptide that likely has greater potential to interact with the coiled-coil trimer. These results provide strong evidence that conserved interhelical packing interactions in the gp41 core are important determinants of HIV-1 entry and its inhibition. These interactions also offer a test-bed for the development of more potent analogues of gp41 peptide inhibitors.     

Structure-function study and anti-HIV activity of synthetic peptide analogues derived from viral chemokine vMIP-II

The viral macrophage inflammatory protein II (vMIP-II) shows a broad spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cellular entry of human immunodeficiency virus type 1 (HIV-1). Recently, we have shown that a synthetic peptide derived from the N-terminus of vMIP-II, designated as V1, is a potent antagonist of CXCR4 but not CCR5 [Zhou, N., et al. (2000) Biochemistry 39, 3782-3787]. In this study, we synthesized a series of new peptides derived from other regions of vMIP-II and characterized their binding activities with both CXCR4 and CCR5. The results provided further support for the notion that the N-terminus of vMIP-II is the major determinant for CXCR4 recognition and that vMIP-II probably interacts with other chemokine receptors such as CCR5 with different sequence and conformational determinants. To understand the structure-function relationship of V1 peptide, its solution conformation was studied using circular dichroism spectroscopy, which showed a random conformation similar to that of the corresponding N-terminus in native vMIP-II. In addition, we synthesized a series of mutant analogues of V1 containing alanine, glycine, or phenylalanine substitution at various positions. Residues Val-1, Arg-7, and Lys-9 of V1 peptide were found to be critical for receptor interaction, because single alanine replacement at these positions dramatically decreased peptide binding to CXCR4. In contrast, alanine or phenylalanine substitution at Cys-11 led to significant enhancement in peptide affinity for CXCR4. Finally, we showed that V1 peptide inhibits HIV-1 replication in CXCR4(+) T-cell lines. These studies provide new insights into the structure-function relationship of V1 peptide and demonstrate that this peptide may be a lead for the development of therapeutic agents.     

Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi's sarcoma-associated herpesvirus at 1.7A

Viral macrophage inflammatory protein I (vMIP-I) is a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) that selectively activates the CC chemokine receptor 8 (CCR8), for which the endogenous ligand is CCL1. The crystal structure of vMIP-I was determined at 1.7A for comparison with other chemokines, especially those that bind CCR8, such as vMIP-II from KSHV, a CCR8 antagonist and the closest homolog (40% identical). vMIP-I has a typical chemokine fold consisting of an extended N-terminal loop, followed by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. The four molecules in the asymmetric unit comprise two MIP-1beta-like dimers. Electrostatic surface representations of CCR8-binding chemokines reveal only minor areas of correlating surface potential, which must be reconciled with promiscuity in receptor and glycosaminoglycan (GAG) binding. In addition, the biological relevance of chemokine oligomerization is examined by comparing the oligomeric states of all chemokine structures deposited to date in the RCSB PDB.     

Crystal structure and structural mechanism of a novel anti-human immunodeficiency virus and D-amino acid-containing chemokine

Chemokines and their receptors play important roles in normal physiological functions and the pathogeneses of a wide range of human diseases, including the entry of human immunodeficiency virus type 1 (HIV-1). However, the use of natural chemokines to probe receptor biology or to develop therapeutic drugs is limited by their lack of selectivity and the poor understanding of mechanisms in ligand-receptor recognition. We addressed these issues by combining chemical and structural biology in research into molecular recognition and inhibitor design. Specifically, the concepts of chemical biology were used to develop synthetically and modularly modified (SMM) chemokines that are unnatural and yet have properties improved over those of natural chemokines in terms of receptor selectivity, affinity, and the ability to explore receptor functions. This was followed by using structural biology to determine the structural basis for synthetically perturbed ligand-receptor selectivity. As a proof-of-principle for this combined chemical and structural-biology approach, we report a novel D-amino acid-containing SMM-chemokine designed based on the natural chemokine called viral macrophage inflammatory protein II (vMIP-II). The incorporation of unnatural D-amino acids enhanced the affinity of this molecule for CXCR4 but significantly diminished that for CCR5 or CCR2, thus yielding much more selective recognition of CXCR4 than wild-type vMIP-II. This D-amino acid-containing chemokine also showed more potent and specific inhibitory activity against HIV-1 entry via CXCR4 than natural chemokines. Furthermore, the high-resolution crystal structure of this D-amino acid-containing chemokine and a molecular-modeling study of its complex with CXCR4 provided the structure-based mechanism for the selective interaction between the ligand and chemokine receptors and the potent anti-HIV activity of D-amino acid-containing chemokines.     

Identification of viral macrophage inflammatory protein (vMIP)-II as a ligand for GPR5/XCR1

Lymphotactin is unique among chemokines in that it contains only two of four conserved cysteines and may possess a structure less constrained than other chemokines. The viral chemokine vMIP-II, which presumably has a structure similar to that of CC chemokines has been shown to inhibit many chemokine receptors, but its activity at GPR5/XCR1 has not been described. Interestingly, vMIP-II (but not vMIP-I) was found to be a potent antagonist of lymphotactin activity at GPR5/XCR1, extending the range of chemokine classes that this viral protein is known to inhibit to include the C class chemokine. In addition, we have extended previous analyses of GPR5/XCR1 expression and show that this receptor is expressed in leukocyte cells previously shown to be responsive to lymphotactin.     

Comparison of the structure of vMIP-II with eotaxin-1, RANTES, and MCP-3 suggests a unique mechanism for CCR3 activation

Herpesvirus-8 macrophage inflammatory protein-II (vMIP-II) binds a uniquely wide spectrum of chemokine receptors. We report the X-ray structure of vMIP-II determined to 2.1 A resolution. Like RANTES, vMIP-II crystallizes as a dimer and displays the conventional chemokine tertiary fold. We have compared the surface topology and electrostatic potential of vMIP-II to those of eotaxin-1, RANTES, and MCP-3, three CCR3 physiological agonists with known three-dimensional structures. Surface epitopes identified on RANTES to be involved in binding to CCR3 are mimicked on the eotaxin-1 and MCP-3 surface. However, the surface topology of vMIP-II in these regions is markedly different. The results presented here indicate that the structural basis for interaction with the chemokine receptor CCR3 by vMIP-II is different from that for the physiological agonists eotaxin-1, RANTES, and MCP-3. These differences on vMIP-II may be a consequence of its broad-range receptor recognition capabilities.     

Complete crystal structure of monocyte chemotactic protein-2, a CC chemokine that interacts with multiple receptors

Monocyte chemotactic protein 2 (MCP-2) is a CC chemokine that utilizes multiple cellular receptors to attract and activate human leukocytes. MCP-2 is a potent inhibitor of HIV-1 by virtue of its high-affinity binding to the receptor CCR5, one of the major coreceptors for HIV-1. Although a few structures of CC chemokines have been reported, none of these was determined with the N-terminal pyroglutamic acid residue (pGlu1) and a complete C-terminus. pGlu1 is essential for the chemotactic activity of MCP-2. Recombinant MCP-2 has Gln1 at the N terminus, 12-15% of which cyclizes automatically and forms pGlu1. The chemotactic activity of such MCP-2 mixture, which contains 12-15% pGlu1-form and 85-88% Gln1-form protein, is approximately 10 times lower when compared with that of fully cyclized MCP-2 preparation. Therefore, this chemokine is practically inactive without pGlu1. We have determined the complete crystal structure of MCP-2 that contains both pGlu1 and an intact C-terminus. With the existence of pGlu1, the conformation of the N-terminus allows two additional interactions between the two subunits of MCP-2 dimer: a hydrogen bond between pGlu1 and Asn17 and a salt bridge between Asp3 and Arg18. Consequently, both pGlu1 are anchored and buried, and thereby, both N-terminal regions are protected against protease degradation. We have also observed not previously reported extended helical nature of the C terminal region, which covers residues 58-74.     

Secondary structure in solution of barwin from barley seed using 1H nuclear magnetic resonance spectroscopy.

Barwin, a basic protein from barley seed of 125 amino acid residues, has been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. This protein is closely related to the C-terminal domain of proteins whose synthesis is induced by wounding, the so-called win proteins. These proteins may, therefore, have a role in the defense against fungal attack. Full assignment of the 1H nuclear magnetic resonances has been obtained for 104 amino acid residues, and 18 amino acid spin systems were partially assigned. Sequence-specific assignment using nuclear Overhauser spectroscopy has been achieved for 122 of the 125 residues. This has revealed that the secondary structure of the protein is dominated by a large four-stranded antiparallel beta-sheet consisting of the strands Gln2-Thr9, Lys65-Asn71, Gln77-Arg81, and His113-Val121, a small parallel beta-sheet of the strands Trp48-Cys52 and Asp84-Ala87, which together account for a third of the protein. Sequential effects indicate the presence of three small alpha-helices, Tyr30-Lys38, Leu40-Tyr46, and Thr97-Asp103. The secondary structure in other regions of the sequence is characterized mainly by loops and turns and regions where no regular secondary structure arrangement could be identified. A large number of long-range nuclear Overhauser effects has been identified, and these have been used, together with sequential and intranuclear Overhauser effects, for a calculation of the protein's three-dimensional structure.     

Crystallographic studies on the role of the C-terminal segment of human angiogenin in defining enzymatic potency

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.     

Solution structure of the capsid protein from the human T-cell leukemia virus type-I.

The solution structure of the capsid protein (CA) from the human T-cell leukemia virus type one (HTLV-I), a retrovirus that causes T-cell leukemia and HTLV-I-associated myelopathy in humans, has been determined by NMR methods. The protein consists of independent N and C-terminal domains connected by a flexible linker. The domains are structurally similar to the N-terminal "core" and C-terminal "dimerization" domains, respectively, of the human immunodeficiency virus type one (HIV-1) and equine infectious anemia virus (EIAV) capsid proteins, although several important differences exist. In particular, hydrophobic residues near the major homology region are partially buried in HTLV-I CA, which is monomeric in solution, whereas analogous residues in HIV-1 and EIAV CA project from the C-terminal domain and promote dimerization. These differences in the structure and oligomerization state of the proteins appear to be related to, and possibly controlled by, the oxidation state of conserved cysteine residues, which are reduced in HTLV-I CA but form a disulfide bond in the HIV-1 and EIAV CA crystal structures. The results are consistent with an oxidative capsid assembly mechanism, in which CA oligomerization or maturation is triggered by disulfide bo nd formation as the budding virus enters the oxidizing environment of the bloodstream.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

Structural reorganization of the copper binding site involving Thr15 of mavicyanin from Cucurbita pepo medullosa (zucchini) upon reduction

Mavicyanin, a glycosylated protein isolated from Cucurbita pepo medullosa (zucchini), is a member of the phytocyanin subfamily containing one polypeptide chain of 109 amino residues and an unusual type-I Cu site in which the copper ligands are His45, Cys86, His91, and Gln96. The crystal structures of oxidized and reduced mavicyanin were determined at 1.6 and 1.9 A resolution, respectively. Mavicyanin has a core structure of seven polypeptide beta-strands arranged as a beta-sandwich organized into two beta-sheets, and the structure considerably resembles that of stellacyanin from cucumber (CST) or cucumber basic protein (CBP). A flexible region was not observed on superimpositioning of the oxidized and reduced mavicyanin structures. However, the Cu(II)-epsilon-O-Gln96 bond length was extended by 0.47 A, and the Thr15 residue was rotated by 60.0 degrees and O-gamma1-Thr15 moved from a distance of 4.78 to 2.58 A from the ligand Gln96 forming a new hydrogen bond between O-gamma1-Thr15 and epsilon-O-Gln96 upon reduction. The reorganization of copper coordination geometry of mavicyanin upon reduction arouses reduction potential decreased above pH 8 [Battistuzzi et al. (2001) J. Inorg. Biochem. 83, 223-227]. The rotation of Thr15 and the hydrogen bonding with the ligand Gln96 may constitute structural evidence of the decrease in the reduction potential at high pH.     

Homology modeling and molecular dynamics simulations of lymphotactin

We have modeled the structure of human lymphotactin (hLpnt), by homology modeling and molecular dynamics simulations. This chemokine is unique in having a single disulfide bond and a long C-terminal tail. Because other structural classes of chemokines have two pairs of Cys residues, compared to one in Lpnt, and because it has been shown that both disulfide bonds are required for stability and function, the question arises how the Lpnt maintains its structural integrity. The initial structure of hLpnt was constructed by homology modeling. The first 63 residues in the monomer of hLpnt were modeled using the structure of the human CC chemokine, RANTES, whose sequence appeared most similar. The structure of the long C-terminal tail, missing in RANTES, was taken from the human muscle fatty-acid binding protein. In a Protein Data Bank search, this protein was found to contain a sequence that was most homologous to the long tail. Consequently, the modeled hLpnt C-terminal tail consisted of both alpha-helical and beta-motifs. The complete model of the hLpnt monomer consisted of two alpha-helices located above the five-stranded beta-sheet. Molecular dynamics simulations of the solvated initial model have indicated that the stability of the predicted fold is related to the geometry of Pro78. The five-stranded beta-sheet appeared to be preserved only when Pro78 was modeled in the cis conformation. Simulations were also performed both for the C-terminal truncated forms of the hLpnt that contained one or two (CC chemokine-like) disulfide bonds, and for the chicken Lpnt (cLpnt). Our MD simulations indicated that the turn region (T30-G34) in hLpnt is important for the interactions with the receptor, and that the long C-terminal region stabilizes both the turn (T30-G34) and the five-stranded beta-sheet. The major conclusion from our theoretical studies is that the lack of one disulfide bond and the extension of the C-terminus in hLptn are mutually complementary. It is very likely that removal of two Cys residues sufficiently destabilizes the structure of a chemokine molecule, particularly the core beta-sheet, to abolish its biological function. However, this situation is rectified by the long C-terminal segment. The role of this long region is most likely to stabilize the first beta-turn region and alpha-helix H1, explaining how this chemokine can function with a single disulfide bond.     

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.