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.     

The crystal structure of mouse Exo70 reveals unique features of the mammalian exocyst

The exocyst is a eukaryotic tethering complex necessary for the fusion of exocytic vesicles with the plasma membrane. Its function in vivo is tightly regulated by interactions with multiple small GTPases. Exo70, one of the eight subunits of the exocyst, is important for the localization of the exocyst to the plasma membrane. It interacts with TC10 and Rho3 GTPases in mammals and yeast, respectively, and has been shown recently to bind to the actin-polymerization complex Arp2/3. Here, we present the crystal structure of Mus musculus Exo70 at 2.25 A resolution. Exo70 is composed of alpha-helices in a series of right-handed helix-turn-helix motifs organized into a long rod of length 170 A and width 35 A. Although the alpha-helical organization of this molecule is similar to that in Saccharomyces cerevisiae Exo70, major structural differences are observed on the surface of the molecule, at the domain boundaries, and in various loop structures. In particular, the C-terminal domain of M. musculus Exo70 adopts a new orientation relative to the N-terminal half not seen in S. cerevisiae Exo70 structures. Given the low level of sequence conservation within Exo70, this structure provides new insights into our understanding of many species-specific functions of the exocyst.     

Structures of the human eIF4E homologous protein, h4EHP, in its m7GTP-bound and unliganded forms

All eukaryotic cellular mRNAs contain a 5' m(7)GpppN cap. In addition to conferring stability to the mRNA, the cap is required for pre-mRNA splicing, nuclear export and translation by providing an anchor point for protein binding. In translation, the interaction between the cap and the eukaryotic initiation factor 4E (eIF4E) is important in the recruitment of the mRNAs to the ribosome. Human 4EHP (h4EHP) is a homologue of eIF4E. Like eIF4E it is able to bind the cap but it appears to play a different cellular role, possibly being involved in the fine-tuning of protein expression levels. Here we use X-ray crystallography and isothermal titration calorimetry (ITC) to investigate further the binding of cap analogues and peptides to h4EHP. m(7)GTP binds to 4EHP 200-fold more weakly than it does to eIF4E with the guanine base sandwiched by a tyrosine and a tryptophan instead of two tryptophan residues as seen in eIF4E. The tyrosine resides on a loop that is longer in h4EHP than in eIF4E. The consequent conformational difference between the proteins allows the tyrosine to mimic the six-membered ring of the tryptophan in eIF4E and adopt an orientation that is similar to that seen for equivalent residues in other non-homologous cap-binding proteins. In the absence of ligand the binding site is incompletely formed with one of the aromatic residues being disordered and the side-chain of the other adopting a novel conformation. A peptide derived from the eIF4E inhibitory protein, 4E-BP1 binds h4EHP 100-fold less strongly than eIF4E but in a similar manner. Overall the data, combined with sequence analyses of 4EHP from evolutionary diverse species, strongly support the hypothesis that 4EHP plays a physiological role utilizing both cap-binding and protein-binding functions but which is distinct from eIF4E.     

Crystal structure of vestitone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids are commonly found in leguminous plants, where they play important roles in plant defense and have significant health benefits for animals and humans. Vestitone reductase catalyzes a stereospecific NADPH-dependent reduction of (3R)-vestitone in the biosynthesis of the antimicrobial isoflavonoid phytoalexin medicarpin. The crystal structure of alfalfa (Medicago sativa L.) vestitone reductase has been determined at 1.4 A resolution. The structure contains a classic Rossmann fold domain in the N terminus and a small C-terminal domain. Sequence and structural analysis showed that vestitone reductase is a member of the short-chain dehydrogenase/reductase (SDR) superfamily despite the low levels of sequence identity, and the prominent structural differences from other SDR enzymes with known structures. The putative binding sites for the co-factor NADPH and the substrate (3R)-vestitone were defined and located in a large cleft formed between the N and C-terminal domains of enzyme. Potential key residues for enzyme activity were also identified, including the catalytic triad Ser129-Tyr164-Lys168. A molecular docking study showed that (3R)-vestitone, but not the (3S) isomer, forms favored interactions with the co-factor and catalytic triad, thus providing an explanation for the enzyme's strict substrate stereo-specificity.