Solution structure of the DNA binding domain of HIV-1 integrase.

The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site.

Sequential backbone assignment of isotopically enriched proteins in D2O by deuterium-decoupled HA(CA)N and HA(CACO)N.

It is demonstrated that sequential resonance assignment of the backbone 1H alpha and 15N resonances of proteins can be obtained without recourse to the backbone amide protons, an approach which should be useful for assignment of regions with rapidly exchanging backbone amide protons and for proteins rich in proline residues. The method relies on the combined use of two 2D experiments, HA(CA)N and HA(CACO)N or their 3D analogs, which correlate 1H alpha with the intraresidue 15N and with the 15N resonance of the next residue. The experiments are preferably conducted in D2O, where very high resolution in the 15N dimension can be achieved by using 2H decoupling. The approach is demonstrated for a sample of human ubiquitin, uniformly enriched in 13C and 15N. Complete backbone and 13C beta/1H beta resonance assignments are presented.

High-resolution solution structure of the beta chemokine hMIP-1 beta by multidimensional NMR.

The three-dimensional structure of a member of the beta subfamily of chemokines, human macrophage inflammatory protein-1 beta (hMIP-1 beta), has been determined with the use of solution multidimensional heteronuclear magnetic resonance spectroscopy. Human MIP-1 beta is a symmetric homodimer with a relative molecular mass of approximately 16 kilodaltons. The structure of the hMIP-1 beta monomer is similar to that of the related alpha chemokine interleukin-8 (IL-8). However, the quaternary structures of the two proteins are entirely distinct, and the dimer interface is formed by a completely different set of residues. Whereas the IL-8 dimer is globular, the hMIP-1 beta dimer is elongated and cylindrical. This provides a rational explanation for the absence of cross-binding and reactivity between the alpha and beta chemokine subfamilies. Calculation of the solvation free energies of dimerization suggests that the formation and stabilization of the two different types of dimers arise from the burial of hydrophobic residues.

Triosephosphate isomerase requires a positively charged active site: the role of lysine-12.

The role of lysine-12 at the active site of yeast triosephosphate isomerase has been elucidated by a combination of site-directed mutagenesis, Fourier transform infrared spectroscopy, enzyme kinetics, and X-ray crystallography. Several lines of evidence suggest that the mutant isomerase in which lysine has been changed to methionine cannot bind substrate. This mutant enzyme has no detectable catalytic activity, and infrared experiments show no evidence of binding dihydroxyacetone phosphate nor dihydroxyacetone sulfate to the active site. Furthermore, crystals of the enzyme grown in the presence of phosphoglycolohydroxamate, a potent reaction intermediate analog, show an open active site with no inhibitor bound. Mutation of lysine-12 to arginine produces a protein with a value Km elevated by a factor of 22, a Vmax reduced by a factor of 180, and a Ki for phosphoglycolohydroxamate elevated by a factor of 290. Mutation of lysine-12 to histidine produces an enzyme that shows virtually no catalytic activity at neutral pH, but below pH 6.1 this enzyme is active, suggesting that protonation of the histidine in this mutant is required for activity. These studies, together with the structural results reported in an accompanying paper, provide convincing evidence that a positive charge is required for substrate binding at the active site of triosephosphate isomerase and that lysine-12 provides this positive charge.

Determination of the secondary structure and folding topology of an RNA binding domain of mammalian hnRNP A1 protein using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure and folding topology of the first RNA binding domain of the human hnRNP A1 protein was determined by multidimensional heteronuclear NMR spectroscopy. The 92 amino acid long domain exhibits a beta alpha beta beta alpha beta folding pattern, arranged in a four-stranded antiparallel beta-sheet flanked by two alpha-helices, which is very similar to that found for other members of this family. Regions of marked variation between the structurally characterized RNA binding proteins of this class to date are mainly localized in the loops connecting the secondary structure elements.

Direct evidence for the exploitation of an alpha-helix in the catalytic mechanism of triosephosphate isomerase.

In previous work, we have shown that the first (and, presumably, the second) pKa of the active-site histidine-95 in triosephosphate isomerase has been lowered by about 2 units [Lodi, P. J., & Knowles, J. R. (1991) Biochemistry 30, 6948-6956]. One reason for the perturbed pKa of this residue appears to be its location at the N-terminus of a short alpha-helix that runs from residues 95 to 102. Fortuitously, the residue at the C-terminus of this helix is also a histidine residue (histidine-103), and the existence of a histidine side chain at each end has allowed us directly to implicate the helix in the perturbation of the pKa value of histidine-95. 15N NMR titration studies of the native enzyme and 13C NMR titration studies of the denatured enzyme show that while the pKa of histidine-95 is lowered by a least 2 units in the folded versus the unfolded state, the pKa of histidine-103 is raised by about 0.6 unit on protein folding. These complementary effects on the pKa values of histidine-95 and histidine-103 suggest that the alpha-helix is indeed responsible for the perturbation of the pKa values. The larger effect on the pKa of histidine-95 is readily rationalized in terms of the local structure of the enzyme. The disparity in the perturbation for the two histidine side chains illustrates how an alpha-helix can be functionally utilized by proteins, directly to affect (as in the present case) the chemistry of catalysis by an enzyme.

Neutral imidazole is the electrophile in the reaction catalyzed by triosephosphate isomerase: structural origins and catalytic implications.

To illuminate the role of histidine-95 in the catalytic reaction mediated by triosephosphate isomerase, 13C and 15N NMR titration studies have been carried out both on the wild-type enzyme and on a mutant isomerase in which the single remaining histidine (that at the active site) has been isotopically enriched in the imidazole ring. 15N NMR has proved especially useful in the unambiguous demonstration that the imidazole ring of histidine-95 is uncharged over the entire pH range of isomerase activity, between pH 5 and pH 9.9. The results require that the first pKa of histidine-95 is below 4.5. This abnormally low pKa rules out the traditional view that the positively charged imidazolium cation of histidine-95 donates a proton to the developing charge on the substrate's carbonyl oxygen. 15N NMR experiments on the enzyme in the presence of the reaction intermediate analogue phosphoglycolohydroxamate show the presence of a strong hydrogen bond between N epsilon 2 of histidine-95 and the bound inhibitor. These findings indicate that, in the catalyzed reaction, proton abstraction from C-1 of dihydroxyacetone phosphate first yields an enediolate intermediate that is strongly hydrogen bonded to the neutral imidazole side chain of histidine-95. The imidazole proton involved in this hydrogen bond then protonates the enediolate, with the transient formation of the enediol-imidazolate ion pair. Abstraction of the hydroxyl proton on O-1 now produces the other enediolate intermediate, which collapses to give the product glyceraldehyde 3-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)