Structure at 2.3 A resolution of the gene 5 product of bacteriophage fd: a DNA unwinding protein.

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been determined by X-ray diffraction analysis of single crystals to 2.3 Å resolution using six isomorphous heavy-atom derivatives. The essentially globular monomer appears to consist of three secondary structural elements, a radically twisted three-stranded antiparallel β sheet and two distinct anti-parallel β loops, which are joined by short segments of extended polypeptide chain. The molecule contains no α-helix. A long groove, or arch, 30 Å in length is formed by the underside of the twisted β sheet and one of the two β ribbons. We believe this groove to be the DNA binding region, and this is supported by the assignment of residues on its surface implicated in binding by solution studies. These residues include several aromatic amino acids which may intercalate or stack upon the bases of the DNA. Two monomers are maintained as a dimer by the very close interaction of symmetry related β ribbons about the molecular dyad. About six residues at the amino and carboxyl terminus are in extended conformation and both seem to exhibit some degree of disorder. The amimo-terminal methionine is the locus for binding the platinum heavy-atom derivatives and tyrosine 26 for attachment of the major iodine substituent.     

A preliminary structure for the DNA binding protein from bacteriophage IKe

A modelling procedure has been utilized to obtain a preliminary three-dimensional structural model for the bacteriophage IKe DNA binding protein (IKe-DBP) based on the known high resolution X-ray diffraction structure of a functionally related protein (G5BP) from bacteriophage fd. The degree of structural homology observed is much higher than the 44% primary sequence identity between these proteins would indicate. These studies suggest IKe-DBP, like G5BP, is composed of a central three-stranded beta sheet from which protrude three extended beta loops. Furthermore, the IKe-DBP structural model can easily form, without conformational rearrangements, the compact dimer unit that is the functionally active species of G5BP. Structural comparisons show residues conserved in the primary sequence of both proteins tend to cluster in two regions. The first being essential for the maintenance of dimer association. The second about the two DNA binding channels which cross the face of each dimer. Based upon an earlier characterized G5BP-DNA complex, a model for DNA complexation to IKe-DBP is also presented.     

Three-dimensional structure of interleukin 8 in solution

The solution structure of the interleukin 8 (IL-8) dimer has been solved by nuclear magnetic resonance (NMR) spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. The structure determination is based on a total of 1880 experimental distance restraints (of which 82 are intersubunit) and 362 torsion angle restraints (comprising phi, psi, and chi 1 torsion angles). A total of 30 simulated annealing structures were calculated, and the atomic rms distribution about the mean coordinate positions (excluding residues 1-5 of each subunit) is 0.41 +/- 0.08 A for the backbone atoms and 0.90 +/- 0.08 A for all atoms. The three-dimensional solution structure of the IL-8 dimer reveals a structural motif in which two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by about 14 A, lie on top of a six-stranded antiparallel beta-sheet platform derived from two three-stranded Greek keys, one from each monomer unit. The general architecture is similar to that of the alpha 1/alpha 2 domains of the human class I histocompatibility antigen HLA-A2. It is suggested that the two alpha-helices form the binding site for the cellular receptor and that the specificity of IL-8, as well as that of a number of related proteins involved in cell-specific chemotaxis, mediation of cell growth, and the inflammatory response, is achieved by the distinct distribution of charged and polar residues at the surface of the helices.     

Structure of the DNA-binding domain of the response regulator PhoP from Mycobacterium tuberculosis

The PhoP-PhoR two-component signaling system from Mycobacterium tuberculosis is essential for the virulence of the tubercle bacillus. The response regulator, PhoP, regulates expression of over 110 genes. In order to elucidate the regulatory mechanism of PhoP, we determined the crystal structure of its DNA-binding domain (PhoPC). PhoPC exhibits a typical fold of the winged helix-turn-helix subfamily of response regulators. The structure starts with a four-stranded antiparallel beta-sheet, followed by a three-helical bundle of alpha-helices, and then a C-terminal beta-hairpin, which together with a short beta-strand between the first and second helices forms a three-stranded antiparallel beta-sheet. Structural elements are packed through a hydrophobic core, with the first helix providing a scaffold for the rest of the domain to pack. The second and third helices and the long, flexible loop between them form the helix-turn-helix motif, with the third helix being the recognition helix. The C-terminal beta-hairpin turn forms the wing motif. The molecular surfaces around the recognition helix and the wing residues show strong positive electrostatic potential, consistent with their roles in DNA binding and nucleotide sequence recognition. The crystal packing of PhoPC gives a hexamer ring, with neighboring molecules interacting in a head-to-tail fashion. This packing interface suggests that PhoPC could bind DNA in a tandem association. However, this mode of DNA binding is likely to be nonspecific because the recognition helix is partially blocked and would be prevented from inserting into the major groove of DNA. Detailed structural analysis and implications with respect to DNA binding are discussed.     

Modeling the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 on the basis of the solution structure of interleukin-8

A model of the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 is presented. The model is predicted based on the previously determined solution structure of interleukin-8 (IL-8/NAP-1) [Clore, G.M., Appella, E., Yamada, M., Matsushima, K. and Gronenborn, A.M. (1990) Biochemistry 29, 1689-1696]. Both proteins belong to a superfamily of cytokine proteins involved in cell-specific chemotaxis, host defense and the inflammatory response. The amino acid sequence identity between the two proteins is 24%. It is shown that the regular secondary structure elements of the parent structure can be retained in the modeled structure, such that the backbone hydrogen bonding pattern is very similar in the two structures. The polypeptide backbone is superimposable with an atomic r.m.s. difference of 0.9 A and all side chains can be modeled by transferring the parent side chain conformation to the new structure. Thus, the deduced structure, like the parent one, is a dimer and consists of a six-stranded antiparallel beta-sheet, formed by two three-stranded Greek keys, one from each monomer, upon which lie two symmetry-related antiparallel alpha-helices, approximately 24 A long and separated by approximately 14 A. All amino acid sequence changes can be accommodated within the parent polypeptide framework without major rearrangements. This is borne out by the fact that the IL-8/NAP-1 and modeled MCAF/MCP-1 structures have similar non-bonding energies. These results strongly suggest that both proteins and all other members of the superfamily most likely have the same tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal Structure of DNA Cytidine Deaminase ABOBEC3G Catalytic Deamination Domain Suggests a Binding Mode of Full-length Enzyme to Single-stranded DNA

APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA.     

Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dimerization of the operator binding domain of phage lambda repressor

Dimerization of lambda repressor is required for its binding to operator DNA. As part of a continuing study of the structural basis of the coupling between dimer formation and operator binding, we have undertaken 1H NMR and gel filtration studies of the dimerization of the N-terminal domain of lambda repressor. Five protein fragments have been studied: three are wild-type fragments of different length (1-102, 1-92, and 1-90), and two are fragments bearing single amino acid substitutions in residues involved in the dimer interface (1-102, Tyr-88----Cys; 1-92, Ile-84----Ser). The tertiary structure of each species is essentially the same, as monitored by the 1H NMR resonances of internal aromatic groups. However, significant differences are observed in their dimerization properties. 1H NMR resonances of aromatic residues that are involved in the dimer contact allow the monomer-dimer equilibrium to be monitored in solution. The structure of the wild-type dimer contact appears to be similar to that deduced from X-ray crystallography and involves the hydrophobic packing of symmetry-related helices (helix 5) from each monomer. Removal of two contact residues, Val-91 and Ser-92, by limited proteolysis disrupts this interaction and also prevents crystallization. The Ile-84----Ser substitution also disrupts this interaction, which accounts for the severely reduced operator affinity of this mutant protein.     

Structure of the PIN/LC8 dimer with a bound peptide.

The structure of the protein known both as neuronal nitric oxide synthase inhibitory protein, PIN (protein inhibitor of nNOS), and also as the 8 kDa dynein light chain (LC8) has been solved by X-ray diffraction. Two PIN/LC8 monomers related by a two-fold axis form a rectangular dimer. Two pairs of alpha-helices cover opposite faces, and each pair of helices packs against a beta-sheet with five antiparallel beta-strands. Each five-stranded beta-sheet contains four strands from one monomer and a fifth strand from the other monomer. A 13-residue peptide from nNOS is bound to the dimer in a deep hydrophobic groove as a sixth antiparallel beta-strand. The structure provides key insights into dimerization of and peptide binding by the multifunctional PIN/LC8 protein.     

The crystal structure of the transcriptional regulator HucR from Deinococcus radiodurans reveals a repressor preconfigured for DNA binding

We report here the 2.3 A resolution structure of the hypothetical uricase regulator (HucR) from Deinococcus radiodurans R1. HucR, a member of the MarR family of DNA-binding proteins, was previously shown to repress its own expression as well as that of a uricase, a repression that is alleviated both in vivo and in vitro upon binding uric acid, the substrate for uricase. As uric acid is a potent scavenger of reactive oxygen species, and as D. radiodurans is known for its remarkable resistance to DNA-damaging agents, these observations indicate a novel oxidative stress response mechanism. The crystal structure of HucR in the absence of ligand or DNA reveals a dimer in which the DNA recognition helices are preconfigured for DNA binding. This configuration of DNA-binding domains is achieved through an apparently stable dimer interface that, in contrast to what is observed in other MarR homologs for which structures have been determined, shows little conformational heterogeneity in the absence of ligand. An additional amino-terminal segment, absent from other MarR homologs, appears to brace the principal helix of the dimerization interface. However, although HucR is preconfigured for DNA binding, the presence of a stacked pair of symmetry-related histidine residues at a central pivot point in the dimer interface suggests a mechanism for a conformational change to attenuate DNA binding.     

Polylysine induces an antiparallel actin dimer that nucleates filament assembly: crystal structure at 3.5-A resolution

An antiparallel actin dimer has been proposed to be an intermediate species during actin filament nucleation. We now show that latrunculin A, a marine natural product that inhibits actin polymerization, arrests polylysine-induced nucleation at the level of an antiparallel dimer, resulting in its accumulation. These dimers, when composed of pyrene-labeled actin subunits, give rise to a fluorescent excimer, permitting detection during polymerization in vitro. We report the crystallographic structure of the polylysine-actin-latrunculin A complex at 3.5-A resolution. The non-crystallographic contact is consistent with a dimeric structure and confirms the antiparallel orientation of its subunits. The crystallographic contacts reveal that the mobile DNase I binding loop of one subunit of a symmetry-related antiparallel actin dimer is partially stabilized in the interface between the two subunits of a second antiparallel dimer. These results provide a potential explanation for the paradoxical nucleation of actin filaments that have exclusively parallel subunits by a dimer containing antiparallel subunits.     

Three-dimensional solution structure of the src homology 2 domain of c-abl.

SH2 regions are protein motifs capable of binding target protein sequences that contain a phosphotyrosine. The solution structure of the abl SH2 product, a protein of 109 residues and 12.1 kd, has been determined by multidimensional nuclear magnetic resonance spectroscopy. It is a compact spherical domain with a pair of three-stranded antiparallel beta sheets and a C-terminal alpha helix enclosing the hydrophobic core. Three arginines project from a short N-terminal alpha helix and one beta sheet into the putative phosphotyrosine-binding site, which lies on a face distal from the termini. Comparison with other SH2 sequences supports a common global fold and mode of phosphotyrosine binding for this family.     

The point mutation A34F causes dimerization of GB1

The immunoglobulin-binding domain B1 of streptococcal protein G (GB1), a very stable, small, single-domain protein, is one of the most extensively used models in the area of protein folding and design. Variants derived from a library of randomized hydrophobic core residues previously revealed alternative folds, namely a completely intertwined tetramer (Frank et al., Nat Struct Biol 2002;9:877-885) and a domain-swapped dimer (Byeon et al., J Mol Biol 2003;333:141-152). Here, we report the NMR structure of the single amino acid mutant Ala-34-Phe which exists as side-by-side dimer. The dimer dissociation constant is 27 +/- 4 microM. The dimer interface comprises two structural elements: First, the beta-sheets of the two monomers pair in an antiparallel arrangement, thereby forming an eight-stranded beta-sheet. Second, the alpha-helix is shortened, ending in a loop that engages in intermolecular contacts. The largest difference between the monomer unit in the A34F dimer and the monomeric wild-type GB1 is the dissolution of the C-terminal half of the alpha-helix associated with a pronounced slow conformational motion of the interface loop. This involves a large movement of the Tyr-33 side chain that swings out from the monomer to engage in dimer contacts.     

Cooperative interactions of the gene 5 protein

Using the refined molecular structure of the Gene 5 DNA Binding Protein (G5BP) and the mechanism of DNA binding deduced from a variety of experimental techniques (G. D. Brayer and A. McPherson, J. Mol. Biol. 169, 565, 1983; G. D. Brayer and A. McPherson, Biochemistry 23, 340, 1984), we have modeled the contiguous, linear aggregation of G5BP dimers along two opposing single strands of DNA. Using both automated graphics systems and systematic calculations of intermolecular contacts between adjacent units, we have optimized the fit of complementary protein surfaces in the presence of DNA. We propose that a minor conformational change involving residues 38-43, triggered by the binding of nucleic acid, relieves several critical steric contacts and permits otherwise extensively complementary surfaces to form an interface. The bonding between surfaces on adjacent G5BP units is the primary source of the cooperativity of binding observed for G5BP. The interacting amino acid residues at the interface are described.