Pulling apart catalytically active Tn5 synaptic complexes using magnetic tweezers

The Tn5 transposase is an example of a class of proteins that move DNA sequences (transposons) via a process called transposition. DNA transposition is a widespread genetic mobility mechanism that has profoundly affected the genomes of nearly all organisms. We have used single-DNA micromanipulation experiments to study the process by which Tn5 DNA transposons are identified and processed by their transposase protein. We have determined that the energy barrier to disassemble catalytically active synaptic complexes is 16 kcal mol(-1). However, we have found that the looping organization of DNA segments by transposase is less sequence-driven than previously thought. Loops anchored at some non-transposon end sequences display a disassembly energy barrier of 14 kcal mol(-1), nearly as stable as the synapses formed at known transposon end sequences. However, these non-transposon end sequence independent complexes do not mediate DNA cleavage. Therefore, the sequence-sensitivity for DNA binding and looping by Tn5 transposase is significantly less than that required for DNA cleavage. These results have implications for the in vivo down regulation of transposition and the cis-transposition bias of transposase.     

Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH

Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.     

Solution structure of the MID1 B-box2 CHC(D/C)C(2)H(2) zinc-binding domain: insights into an evolutionarily conserved RING fold

The B-box type 2 domain is a prominent feature of a large and growing family of RING, B-box, coiled-coil (RBCC) domain-containing proteins and is also present in more than 1500 additional proteins. Most proteins usually contain a single B-box2 domain, although some proteins contain tandem domains consisting of both type 1 and type 2 B-boxes, which actually share little sequence similarity. Recently, we determined the solution structure of B-box1 from MID1, a putative E3 ubiquitin ligase that is mutated in X-linked Opitz G/BBB syndrome, and showed that it adopted a betabetaalpha RING-like fold. Here, we report the tertiary structure of the B-box2 (CHC(D/C)C(2)H(2)) domain from MID1 using multidimensional NMR spectroscopy. This MID1 B-box2 domain consists of a short alpha-helix and a structured loop with two short anti-parallel beta-strands and adopts a tertiary structure similar to the B-box1 and RING structures, even though there is minimal primary sequence similarity between these domains. By mutagenesis, ESI-FTICR and ICP mass spectrometry, we show that the B-box2 domain coordinates two zinc atoms with a 'cross-brace' pattern: one by Cys175, His178, Cys195 and Cys198 and the other by Cys187, Asp190, His204, and His207. Interestingly, this is the first case that an aspartic acid is involved in zinc atom coordination in a zinc-finger domain, although aspartic acid has been shown to coordinate non-catalytic zinc in matrix metalloproteinases. In addition, the finding of a Cys195Phe substitution identified in a patient with X-linked Opitz GBBB syndrome supports the importance of proper zinc coordination for the function of the MID1 B-box2 domain. Notably, however, our structure differs from the only other published B-box2 structure, that from XNF7, which was shown to coordinate one zinc atom. Finally, the similarity in tertiary structures of the B-box2, B-box1 and RING domains suggests these domains have evolved from a common ancestor.     

The structure of the Alzheimer amyloid beta 10-35 peptide probed through replica-exchange molecular dynamics simulations in explicit solvent

The conformational states sampled by the Alzheimer amyloid beta (10-35) (Abeta 10-35) peptide were probed using replica-exchange molecular dynamics (REMD) simulations in explicit solvent. The Abeta 10-35 peptide is a fragment of the full-length Abeta 40/42 peptide that possesses many of the amyloidogenic properties of its full-length counterpart. Under physiological temperature and pressure, our simulations reveal that the Abeta 10-35 peptide does not possess a single unique folded state. Rather, this peptide exists as a mixture of collapsed globular states that remain in rapid dynamic equilibrium with each other. This conformational ensemble is dominated by random coil and bend structures with insignificant presence of an alpha-helical or beta-sheet structure. The 3D structure of Abeta 10-35 is seen to be defined by a salt bridge formed between the side-chains of K28 and D23. This salt bridge is also observed in Abeta fibrils and our simulations suggest that monomeric conformations of Abeta 10-35 contain pre-folded structural motifs that promote rapid aggregation of this peptide.     

Calculation of pKas in RNA: on the structural origins and functional roles of protonated nucleotides

pK(a) calculations based on the Poisson-Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK(a) shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK(a) shifts of nucleotides in RNA structures using numerical solutions to the Poisson-Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK(a)s of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK(a)s: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions.     

Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.     

Crystal structures of Trypanosoma brucei and Staphylococcus aureus mevalonate diphosphate decarboxylase inform on the determinants of specificity and reactivity

Mevalonate diphosphate decarboxylase (MDD) catalyzes the ATP-dependent decarboxylation of mevalonate 5-diphosphate (MDP) to form isopentenyl pyrophosphate, a ubiquitous precursor for isoprenoid biosynthesis. MDD is a poorly understood component of this important metabolic pathway. Complementation of a temperature-sensitive yeast mutant by the putative mdd genes of Trypanosoma brucei and Staphylococcus aureus provides proof-of-function. Crystal structures of MDD from T. brucei (TbMDD, at 1.8 A resolution) and S. aureus (SaMDD, in two distinct crystal forms, each diffracting to 2.3 A resolution) have been determined. Gel-filtration chromatography and analytical ultracentrifugation experiments indicate that TbMDD is predominantly monomeric in solution while SaMDD is dimeric. The new crystal structures and comparison with that of the yeast Saccharomyces cerevisiae enzyme (ScMDD) reveal the structural basis for this variance in quaternary structure. The presence of an ordered sulfate in the structure of TbMDD reveals for the first time details of a ligand binding in the MDD active site and, in conjunction with well-ordered water molecules, comparisons with the related enzyme mevalonate kinase, structural and biochemical data derived on ScMDD and SaMDD, allows us to model a ternary complex with MDP and ATP. This model facilitates discussion of the molecular determinants of substrate recognition and contributions made by specific residues to the enzyme mechanism.     

Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: a glycosylated protein in a sulphate-reducing bacterium

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 A resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane.     

Design of disulfide-linked thioredoxin dimers and multimers through analysis of crystal contacts

Disulfide bonds play an important role in protein stability and function. Here, we describe a general procedure for generating disulfide-linked dimers and multimers of proteins of known crystal structures. An algorithm was developed to predict sites in a protein compatible with intermolecular disulfide formation with neighboring molecules in the crystal lattice. A database analysis was carried out on 46 PDB coordinates to verify the general applicability of this algorithm to predict intermolecular disulfide linkages. On the basis of the predictions from this algorithm, mutants were constructed and characterized for a model protein, thioredoxin. Of the five mutants, as predicted, in solution four formed disulfide-linked dimers while one formed polymers. Thermal and chemical denaturation studies on these mutant thioredoxins showed that three of the four dimeric mutants had similar stability to wild-type thioredoxin while one had lower stability. Three of the mutant dimers crystallized readily (in four to seven days) in contrast to the wild-type protein, which is particularly difficult to crystallize and takes more than a month to form diffraction-quality crystals. In two of the three cases, the structure of the dimer was exactly as predicted by the algorithm, while in the third case the relative orientation of the monomers in the dimer was different from the predicted one. This methodology can be used to enhance protein crystallizability, modulate the oligomerization state and to produce linear chains or ordered three-dimensional protein arrays.     

The crystal structure of the extracellular domain of the inhibitor receptor expressed on myeloid cells IREM-1

The immune receptors expressed on myeloid cells (IREM) are type I transmembrane proteins encoded on human chromosome 17 (17q25.1), whose function is believed to be important in controlling inflammation. To date, three IREM receptors have been identified. IREM-1 functions as an inhibitory receptor, whereas IREM-2 and IREM-3 serve an activating function. Here, we report the crystal structure of IREM-1 extracellular domain at 2.6 A resolution. The overall fold of IREM-1 resembles that of a V-type immunoglobulin domain, and reveals overall close homology with immunoglobulin domains from other immunoreceptors such as CLM-1, TREM-1, TLT-1 and NKp44. Comparing the surface electrostatic potential and hydrophobicity of IREM-1 with its murine homologous CLM-1, we observed unique structural properties for the complementary determining region of IREM-1, which suggests that they may be involved in recognition of the IREM-1 ligand. Particularly interesting is the structural conformation and physical properties of the antibody's equivalent CDR3 loop, which we show to be a structurally variable region of the molecule and therefore could be the main structural determinant for ligand discrimination and binding. In addition, the analysis of the IREM-1 structure revealed the presence of four structurally different cavities. Three of these cavities form a continuous hydrophobic groove on the IREM-1 surface, which point to a region of the molecule capable of accommodating potential ligands.