All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys‐1His array (CCHC = C‐X2‐C‐X4‐H‐X4‐C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc‐binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc‐edge extended X‐ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus‐bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840–847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys‐1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV‐1 NC protein and have determined its three‐dimensional solution‐state structure. The CCHC arrays of HIV‐1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV‐1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid‐binding results provide a basis for the rational design of new NC‐targeted antiviral agents and vaccines for the control of AIDS.

Macrophage migration inhibitory factor (MIF) is an important immunoregulatory molecule with a unique ability to suppress the anti-inflammatory effects of glucocorticoids. Although considered a cytokine, MIF possesses a three-dimensional structure and active site similar to those of 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase. Moreover, a number of catalytic activities have been defined for MIF. To gain insight into the role of catalysis in the biological function of MIF, we have begun to characterize the catalytic activities in more detail. Here we report the crystal structure of MIF complexed with p-hydroxyphenylpyruvate, a substrate for the phenylpyruvate tautomerase activity of MIF. The three binding sites for p-hydroxyphenylpyruvate in the MIF trimer lie at the interface between two subunits. The substrate interacts with Pro-1, Lys-32, and Ile-64 from one subunit and Tyr-95 and Asn-97 from an adjacent subunit. Pro-1 is positioned to function as a catalytic base. There is no functional group that polarizes the alpha-carbonyl of the substrate to weaken the adjacent C-H bond. Mutation of Pro-1 to glycine substantially reduces the catalytic activity. The insertion of an alanine between Pro-1 and Met-2 essentially abolishes activity. Structural studies of these mutants define a source of the reduced activity and provide insight into the mechanism of the catalytic reaction.

Macrophage migration inhibitory factor (MIF) is a secreted protein that activates macrophages, neutrophils and T cells, and is implicated in sepsis, adult respiratory distress syndrome and rheumatoid arthritis. The mechanism of MIF function, however, is unknown. The three-dimensional structure of MIF is unlike that of any other cytokine, but bears striking resemblance to three microbial enzymes, two of which possess an N-terminal proline that serves as a catalytic base. Human MIF also possesses an N-terminal proline (Pro-1) that is invariant among all known homologues. Multiple sequence alignment of these MIF homologues reveals additional invariant residues that span the entire polypeptide but are in close proximity to the N-terminal proline in the folded protein. We find that p-hydroxyphenylpyruvate, a catalytic substrate of MIF, binds to the N-terminal region and interacts with Pro-1. Mutation of Pro-1 to a glycine substantially reduces the catalytic and cytokine activity of MIF. We suggest that the underlying biological activity of MIF may be based on an enzymatic reaction. The identification of the active site should facilitate the development of structure-based inhibitors.

The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)

The three-dimensional solution-state structure is reported for the zinc-substituted form of rubredoxin (Rd) from the marine hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 " c . Structures were generated with DSPACEB by a hybrid distance geometry (DG)-based simulated annealing (SA) approach that employed 403 nuclear Overhauser effect (N0E)-derived interproton distance restraints, including 67 interresidue, 124 sequential ( i -j = I), 75 medium-range ( i -j = 2-5), and 137 long-range ( i -j > 5) restraints.All lower interproton distance bounds were set at the sum of the van Der Waals radii (1.8 A), and upper bounds of 2.7 A, 3.3 A, and 5.0 A were employed to represent qualitatively observed strong, medium, and weak NOE cross peak intensities, respectively. Twenty-three backbone-backbone, six backbone-sulfur (Cys), two backboneside chain, and two side chain-side chain hydrogen bond restraints were include for structure refinement, yielding a total of 436 nonbonded restraints, which averages to >16 restraints per residue. A total of 10 structures generated from random atom positions and 30 structures generated by molecular replacement using the backbone coordinates of Clostridiumpusteuriunum Rd converged to a common conformation, with the average penalty (= sum of the square of the distance bounds violations; +standard deviation) of 0.024 f 0.003 A 2 and a maximum total penalty of 0.035 A*. Superposition of the backbone atoms (C, Ca, N) of residues A1-L51 for all 40 structures afforded an average pairwise root mean square (rms) deviation value (fSD) of 0.42 f 0.07 A. Superposition of all heavy atoms for residues AI-L51, including those of structurally undefined external side chains, afforded an average pairwise rms deviation of 0.72 k 0.08 A . Qualitative comparison of back-calculated and experimental two-dimensional NOESY spectra indicate that the DG/SA structures are consistent with the experimental spectra. The global folding of P. furiosus Zn(Rd) is remarkably similar to the folding observed by X-ray crystallography for native Rd from the mesophilic organism C. pusteuriunum, with the average rms deviation value for backbone atoms of residues Al-LSI of P. furiosus Zn(Rd) superposed with respect to residues K2-V52 of C. pusteuriunum Rd of 0.77 +_ 0.06 A. The conformations of aromatic residues that compose the hydrophobic cores of the two proteins are also similar. However, P. furiosus Rd contains several unique structural elements, including at least four additional hydrogen bonds and three potential electrostatic interactions. Four of these interactions involve the nonconservatively substituted Glu 14, Ala 1, and Trp 3 residues. The combined findings are consistent with the proposal that stabilization of the N-terminal residues inhibits the &sheet from "unzipping" at elevated temperatures (Blake, P.R., Park, J.-B., Bryant, EO., et al., 1991, Biochemistry30, 10885-10895). In view of the high structural similarities between this hyperthermophilic protein and C...

The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.

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