Molecular mechanism underlying the interaction of typical Sac10b family proteins with DNA

The Sac10b protein family is regarded as a family of DNA-binding proteins that is highly conserved and widely distributed within the archaea. Sac10b family members are typically small basic dimeric proteins that bind to DNA with cooperativity and no sequence specificity and are capable of constraining DNA negative supercoils, protecting DNA from Dnase I digestion, and do not compact DNA obviously. However, a detailed understanding of the structural basis of the interaction of Sac10b family proteins with DNA is still lacking. Here, we determined the crystal structure of Mth10b, an atypical member of the Sac10b family from Methanobacterium thermoautotrophicum ΔH, at 2.2 Å. Unlike typical Sac10b family proteins, Mth10b is an acidic protein and binds to neither DNA nor RNA. The overall structure of Mth10b displays high similarity to its homologs, but three pairs of conserved positively charged residues located at the presumed DNA-binding surface are substituted by non-charged residues in Mth10b. Through amino acids interchanges, the DNA-binding ability of Mth10b was restored successfully, whereas the DNA-binding ability of Sso10b, a typical Sac10b family member, was weakened greatly. Based on these results, we propose a model describing the molecular mechanism underlying the interactions of typical Sac10b family proteins with DNA that explains all the characteristics of the interactions between typical Sac10b family members and DNA.     

Biochemical and Structural Insights into RNA Binding by Ssh10b,a Member of the Highly Conserved Sac10b Protein Family in Archaea

Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular β-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.     

Crystal structure of archaeal chromatin protein Alba2-double-stranded DNA complex from Aeropyrum pernix K1

All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.     

Solution structure, stability, and nucleic acid binding of the hyperthermophile protein Sso10b2

The Sso10b (or Alba) family of proteins is a conserved group of archaeal and eukaryotic proteins which are thought to play a role in both chromatin organization and RNA metabolism. We describe here the solution structure and properties of Sso10b2 from Sulfolobus solfataricus. NMR data including residual dipolar couplings and (15)N relaxation data demonstrated that the protein adopts a beta(1)alpha(1)beta(2)alpha(2)beta(3)beta(4) topology with an IF-3-like fold. The protein dimerizes in solution at 30 degrees C via a hydrophobic surface defined by the C-terminal alpha(2)beta(3)beta(4) elements with a structure similar to one of the putative dimers indicated by previous crystal structures. DSC and circular dichroism data demonstrated an unusual two-state structural transition near the growth temperature which led to an increase in beta-sheet content without dissociation of the dimer. The cooperativity of the transition exceeded that of a dimer at pH 7, demonstrating the presence of higher order oligomers near the growth temperature at pH 7. Reverse titrations of Sso10b2 with nucleic acid showed that the protein binds single-stranded DNA (K(d) of 3 x 10(-)(7) M) with higher affinity than RNA (1.3 x 10(-)(6) M) or double-stranded DNA (1.5 x 10(-)(5) M) in 10 mM KH(2)PO(4) (pH 7.0, 20 degrees C). NMR chemical shift perturbation data indicated that single-stranded DNA and RNA binding occurred across the same dimer interface and encompassed a surface defined by the C-terminal ends of the beta(1), beta(2), and beta(3) strands of each monomer.     

The 2.2 A crystal structure of Hsp33: a heat shock protein with redox-regulated chaperone activity

BACKGROUND: One strategy that cells employ to respond to environmental stresses (temperature, oxidation, and pathogens) is to increase the expression of heat shock proteins necessary to maintain viability. Several heat shock proteins function as molecular chaperones by binding unfolded polypeptides and preventing their irreversible aggregation. Hsp33, a highly conserved bacterial heat shock protein, is a redox-regulated molecular chaperone that appears to protect cells against the lethal effects of oxidative stress. RESULTS: The 2.2 A crystal structure of a truncated E. coli Hsp33 (residues 1-255) reveals a domain-swapped dimer. The core domain of each monomer (1-178) folds with a central helix that is sandwiched between two beta sheets. The carboxyl-terminal region (179-235), which lacks the intact Zn binding domain of Hsp33, folds into three helices that pack on the other subunit. The interface between the two core domains is comprised of conserved residues, including a rare Glu-Glu hydrogen bond across the dyad axis. Two potential polypeptide binding sites that span the dimer are observed: a long groove containing pockets of conserved and hydrophobic residues, and an intersubunit 10-stranded beta sheet "saddle" with a largely uncharged or hydrophobic surface. CONCLUSIONS: Hsp33 is a dimer in the crystal structure. Solution studies confirmed that this dimer reflects the structural changes that occur upon activation of Hsp33 as a molecular chaperone. Patterns of conserved residues and surface charges suggest that two grooves might be potential binding sites for protein folding intermediates.     

Crystal structure of the hyperthermophilic archaeal DNA-binding protein Sso10b2 at a resolution of 1.85 Angstroms

The crystal structure of a small, basic DNA binding protein, Sso10b2, from the thermoacidophilic archaeon Sulfolobus solfataricus was determined by the Zn multiwavelength anomalous diffraction method and refined to 1.85 A resolution. The 89-amino-acid protein adopts a betaalphabetaalphabetabeta topology. The structure is similar to that of Sso10b1 (also called Alba) from the same organism. However, Sso10b2 contains an arginine-rich loop RDRRR motif, which may play an important role in nucleic acid binding. There are two independent Sso10b2 proteins in the asymmetric unit, and a plausible stable dimer could be deduced from the crystal structure. Topology comparison revealed that Sso10b2 is similar to several RNA-binding proteins, including IF3-C, YhhP, and DNase I. Models of the Sso10b2 dimer bound to either B-DNA or A-DNA have been constructed.     

Two conformations of archaeal Ssh10b. The origin of its temperature-dependent interaction with DNA

The DNA-binding protein Ssh10b from the hyperthermophilic archaeon Sulfolobus shibatae is a member of the Sac10b family, which has been speculated to be involved in the organization of the chromosomal DNA in Archaea. Ssh10b affects the DNA topology in a temperature dependent fashion that has not been reported for any other DNA-binding proteins. Heteronuclear NMR and site-directed mutagenesis were used to analyze the structural basis of the temperature-dependent Ssh10b-DNA interaction. The data analysis indicates that two forms of Ssh10b homodimers co-exist in solution, and the slow cis-trans isomerization of the Leu61-Pro62 peptide bond is the key factor responsible for the conformational heterogeneity of the Ssh10b homodimer. The T-form dimer, with the Leu61-Pro62 bond in the trans conformation, dominates at higher temperature, whereas population of the C-form dimer, with the bond in the cis conformation, increases on decreasing the temperature. The two forms of the Ssh10b dimer show the same DNA binding site but have different conformational features that are responsible for the temperature-dependent nature of the Ssh10b-DNA interaction.     

Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase

Comparative structural studies on proteins derived from organisms with growth optima ranging from 15 to 100 degrees C are beginning to shed light on the mechanisms of protein thermoadaptation. One means of sustaining hyperthermostability is for proteins to exist in higher oligomeric forms than their mesophilic homologues. Triosephosphate isomerase (TIM) is one of the most studied enzymes, whose fold represents one of nature's most common protein architectures. Most TIMs are dimers of approximately 250 amino acid residues per monomer. Here, we report the 2.7 A resolution crystal structure of the extremely thermostable TIM from Pyrococcus woesei, a hyperthermophilic archaeon growing optimally at 100 degrees C, representing the first archaeal TIM structure. P. woesei TIM exists as a tetramer comprising monomers of only 228 amino acid residues. Structural comparisons with other less thermostable TIMs show that although the central beta-barrel is largely conserved, severe pruning of several helices and truncation of some loops give rise to a much more compact monomer in the small hyperthermophilic TIM. The classical TIM dimer formation is conserved in P. woesei TIM. The extreme thermostability of PwTIM appears to be achieved by the creation of a compact tetramer where two classical TIM dimers interact via an extensive hydrophobic interface. The tetramer is formed through largely hydrophobic interactions between some of the pruned helical regions. The equivalent helical regions in less thermostable dimeric TIMs represent regions of high average temperature factor. The PwTIM seems to have removed these regions of potential instability in the formation of the tetramer. This study of PwTIM provides further support for the role of higher oligomerisation states in extreme thermal stabilisation.     

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution

The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase has been determined at 2.7 A resolution. Its first 120 amino acids form a central five-stranded, beta-pleated sheet surrounded by five alpha helices. In one of the four dyad-related dimers, the two active site Ser-10 residues are 19 A apart, perhaps close enough to contact and become covalently linked to the DNA at the recombination site. This dimer also forms the only closely packed tetramer found in the crystal. The subunit interface at a second dyad-related dimer is more extensive and more highly conserved among the homologous recombinases; however, its active site Ser-10 residues are more than 30 A apart. Side chains, identified by mutations that eliminate catalysis but not DNA binding, are located on the subunit surface near the active site serine and at the interface between a third dyad-related pair of subunits of the tetramer.     

Crystal structure of DNA gyrase B′ domain sheds lights on the mechanism for T-segment navigation

DNA gyrase is an indispensible marvelous molecular machine in manipulating the DNA topology for the prokaryotes. In the ‘two-gate’ mechanism of DNA topoisomerase, T-segment navigation from N- to DNA-gate is a critical step, but the structural basis supporting this scheme is unclear. The crystal structure of DNA gyrase B′ subfragment from Mycobacterium tuberculosis reveals an intrinsic homodimer. The two subunits, each consisting of a Tail and a Toprim domain, are tightly packed one another to form a ‘crab-like’ organization never observed previously from yeast topo II. Structural comparisons show two orientational alterations of the Tail domain, which may be dominated by a 43-residue peptide at the B′ module C-terminus. A highly conserved pentapeptide mediates large-scale intrasubunit conformational change as a hinge point. Mutational studies highlight the significant roles of a negatively charge cluster on a groove at dimer interface. On the basis of structural analysis and mutation experiments, a sluice-like model for T-segment transport is proposed.     

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A₂ homologue: insights into structural determinants for myotoxicity and dimeric configuration

Catalytically inactive phospholipase A(2) (PLA(2)) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA(2) homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA(2) homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA(2) homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement.     

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.     

Crystal structure of an Xrcc4-DNA ligase IV complex.

A complex of two proteins, Xrcc4 and DNA ligase IV, plays a fundamental role in DNA non-homologous end joining (NHEJ), a cellular function required for double-strand break repair and V(D)J recombination. Here we report the crystal structure of human Xrcc4 bound to a polypeptide that corresponds to the DNA ligase IV sequence linking its two BRCA1 C-terminal (BRCT) domains. In the complex, a single ligase chain binds asymmetrically to an Xrcc4 dimer. The helical tails of Xrcc4 undergo a substantial conformational change relative to the uncomplexed protein, forming a coiled coil that unwinds upon ligase binding, leading to a flat interaction surface. A buried network of charged hydrogen bonds surrounded by extensive hydrophobic contacts explains the observed tightness of the interaction. The strong conservation of residues at the interface between the two proteins provides evidence that the observed mode of interaction has been maintained in NHEJ throughout evolution.     

Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State

Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.     

Mth10b, a unique member of the Sac10b family, does not bind nucleic acid

The Sac10b protein family is regarded as a group of nucleic acid-binding proteins that are highly conserved and widely distributed within archaea. All reported members of this family are basic proteins that exist as homodimers in solution and bind to DNA and/or RNA without apparent sequence specificity in vitro. Here, we reported a unique member of the family, Mth10b from Methanobacterium thermoautotrophicum ΔH, whose amino acid sequence shares high homology with other Sac10b family proteins. However, unlike those proteins, Mth10b is an acidic protein; its potential isoelectric point is only 4.56, which is inconsistent with the characteristics of a nucleic acid-binding protein. In this study, Mth10b was expressed in Escherichia coli and purified using a three-column chromatography purification procedure. Biochemical characterization indicated that Mth10b should be similar to typical Sac10b family proteins with respect to its secondary and tertiary structure and in its preferred oligomeric forms. However, an electrophoretic mobility shift analysis (EMSA) showed that neither DNA nor RNA bound to Mth10b in vitro, indicating that either Mth10b likely has a physiological function that is distinct from those of other Sac10b family members or nucleic acid-binding ability may not be a fundamental factor to the actual function of the Sac10b family.