The crystal structure of benzyloxycarbonyl-(α-aminoisobutyryl)-L-alanyl methyl ester

Crystals of the title compound, C20,H29,N3,O6, are monoclinic, space group P2, with a = 8-839 (3), b = f10.818 (3), c = 11.414 (2) A, β = 95.69 (2)° Z = 2; final R = 0.053. The molecular conformation is defined by the following angles (φ, ψ): Aib-1 58- 1, 36.8; Aib-2 68.3, 18.6; Ala-3 (φ) -136.2°. The molecule adopts a type 111 β -turn conformation stabilized by an intramolecular hydrogen bond between the CO of the benzyloxycarbonyl group and the NH of the alanyl residue. The hydrogen-bond parameters are N···O 2-904 Å and ∠NH···O 156.9°

Molecular structure of Boc-Aib-Aib-Phe-Met-NH<SUB>2</SUB>·DMSO. A fragment of a biologically active enkephalin analogue

The tetrapeptide t-butyloxycarbonyl-&#945;-aminoisobutyryl-&#945;-aminoisobutyryl-L- phenylalanyl-L-methionyl amide crystallizes in the orthorhombic space group P212121 with a= 9.096, b= 18.067, c= 21.701 &#197; and Z= 4. The crystals contain one molecule of dimethyl sulphoxide (DMSO) associated with each peptide. The structure has been solved by direct methods and refined to an R value of 0.103 for 2 672 observed reflections. The peptide adopts a distorted 310 helical structure stabilized by two intramolecular 4 &#8594; 1 hydrogen bonds between the Boc CO and Aib(1) CO groups and the NH groups of Phe(3) and Met(4), respectively. A long hydrogen bond (N...O = 3.35 &#197;) is also observed between Aib(2) CO and one of the terminal amide hydrogens. The DMSO molecule is strongly hydrogen bonded to the Aib(1) NH group. The solid-state conformation agrees well with proposals made on the basis of n.m.r. studies in solution

A New Crucial Protein Interaction Element That Targets the Adenovirus E4-ORF1 Oncoprotein to Membrane Vesicles

Human adenovirus type 9 exclusively elicits mammary tumors in experimental animals, and the primary oncogenic determinant of this virus is the E4-ORF1 oncogene, as opposed to the well-known E1A and E1Boncogenes. The tumorigenic potential of E4-ORF1, as well as its ability to oncogenically stimulate phosphatidylinositol 3-kinase (PI3K), depends on a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with several different membrane-associated cellular PDZ proteins, including MUPP1, PATJ, MAGI-1, ZO-2, and Dlg1. Nevertheless, because certain E4-ORF1 mutations that alter neither the sequence nor the function of the PBM abolish E4-ORF1-induced PI3K activation and cellular transformation, we reasoned that E4-ORF1 must possess an additional crucial protein element. In the present study, we identified seven E4-ORF1 amino acid residues that define this new element, designated domain 2, and showed that it mediates binding to a 70-kDa cellular phosphoprotein. We also discovered that domain 2 or the PBM independently promotes E4-ORF1 localization to cytoplasmic membrane vesicles and that this activity of domain 2 depends on E4-ORF1 trimerization. Consistent with the latter observation, molecular-modeling analyses predicted that E4-ORF1 trimerization brings together six out of seven domain 2 residues at each of the three subunit interfaces. These findings importantly demonstrate that PI3K activation and cellular transformation induced by E4-ORF1 require two separate protein interaction elements, domain 2 and the PBM, each of which targets E4-ORF1 to vesicle membranes in cells

Functionally distinct monomers and trimers produced by a viral oncoprotein

While the process of homo-oligomer formation and disassembly into subunits represents a common strategy to regulate protein activity, reports of proteins in which the subunit and homo-oligomer perform independent functions are scarce. Tumorigenesis induced by the adenovirus E4-ORF1 oncoprotein depends on its binding to a select group of cellular PDZ proteins, including MUPP1, MAGI-1, ZO-2 and Dlg1. We report here that in cells E4-ORF1 exists as both a monomer and trimer and that monomers specifically bind and sequester MUPP1, MAGI-1 and ZO-2 within insoluble complexes whereas trimers specifically bind Dlg1 and promote its translocation to the plasma membrane. This work exposes a novel strategy wherein the oligomerization state of a protein not only determines the capacity to bind separate related targets but also couples the interactions to different functional consequences

Structural basis for different substrate profiles of two closely related class D β-lactamases and their inhibition by halogens

OXA-163 and OXA-48 are closely related class D β-lactamases that exhibit different substrate profiles. OXA-163 hydrolyzes oxyimino-cephalosporins, particularly ceftazidime, while OXA-48 prefers carbapenem substrates. OXA-163 differs from OXA-48 by one substitution (S212D) in the active-site β5 strand and a four-amino acid deletion (214-RIEP-217) in the loop connecting the β5 and β6 strands. Although the structure of OXA-48 has been determined, the structure of OXA-163 is unknown. To further understand the basis for their different substrate specificities, we performed enzyme kinetic analysis, inhibition assays, X-ray crystallography, and molecular modeling. The results confirm the carbapenemase nature of OXA-48 and the ability of OXA-163 to hydrolyze the oxyimino-cephalosporin ceftazidime. The crystal structure of OXA-163 determined at 1.72 Å resolution reveals an expanded active site compared to that of OXA-48, which allows the bulky substrate ceftazidime to be accommodated. The structural differences with OXA-48, which cannot hydrolyze ceftazidime, provide a rationale for the change in substrate specificity between the enzymes. OXA-163 also crystallized under another condition that included iodide. The crystal structure determined at 2.87 Å resolution revealed iodide in the active site accompanied by several significant conformational changes, including a distortion of the β5 strand, decarboxylation of Lys73, and distortion of the substrate-binding site. Further studies showed that both OXA-163 and OXA-48 are inhibited in the presence of iodide. In addition, OXA-10, which is not a member of the OXA-48-like family, is also inhibited by iodide. These findings provide a molecular basis for the hydrolysis of ceftazidime by OXA-163 and, more broadly, show how minor sequence changes can profoundly alter the active-site configuration and thereby affect the substrate profile of an enzyme

Antagonism between substitutions in β-lactamase explains a path not taken in the evolution of bacterial drug resistance

CTX-M β-lactamases are widespread in Gram-negative bacterial pathogens and provide resistance to the cephalosporin cefotaxime but not to the related antibiotic ceftazidime. Nevertheless, variants have emerged that confer resistance to ceftazidime. Two natural mutations, causing P167S and D240G substitutions in the CTX-M enzyme, result in 10-fold increased hydrolysis of ceftazidime. Although the combination of these mutations would be predicted to increase ceftazidime hydrolysis further, the P167S/D240G combination has not been observed in a naturally occurring CTX-M variant. Here, using recombinantly expressed enzymes, minimum inhibitory concentration measurements, steady-state enzyme kinetics, and X-ray crystallography, we show that the P167S/D240G double mutant enzyme exhibits decreased ceftazidime hydrolysis, lower thermostability, and decreased protein expression levels compared with each of the single mutants, indicating negative epistasis. X-ray structures of mutant enzymes with covalently trapped ceftazidime suggested that a change of an active-site Ω-loop to an open conformation accommodates ceftazidime leading to enhanced catalysis. 10-μs molecular dynamics simulations further correlated Ω-loop opening with catalytic activity. We observed that the WT and P167S/D240G variant with acylated ceftazidime both favor a closed conformation not conducive for catalysis. In contrast, the single substitutions dramatically increased the probability of open conformations. We conclude that the antagonism is due to restricting the conformation of the Ω-loop. These results reveal the importance of conformational heterogeneity of active-site loops in controlling catalytic activity and directing evolutionary trajectories

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.Instituto de VirologíaFil: Salmen, Wilhelm. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Hu, Liya. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnologicas; ArgentinaFil: Bok, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chaimongkol, Natthawan. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Caliciviruses Section; Estados UnidosFil: Ettayebi, Khalil. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Sosnovtsev, Stanislav V. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Caliciviruses Section; Estados UnidosFil: Soni, Kaundal. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Ayyar, B. Vijayalakshmi. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Shanker, Sreejesh. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Neill, Frederick H. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Sankaran, Banumathi. Berkeley Center for Structural Biology. Molecular Biophysics and Integrated Bioimaging. Lawrence Berkeley Laboratory; Estados UnidosFil: Atmar, Robert L. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Atmar, Robert L. Baylor College of Medicine. Department of Medicine; Estados UnidosFil: Estes, Mary K. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados UnidosFil: Estes, Mary K. Baylor College of Medicine. Department of Medicine; Estados UnidosFil: Green, Kim Y. National Institutes of Health. National Institute of Allergy and Infectious Diseases. Caliciviruses Section; Estados UnidosFil: Parreño, Gladys Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virologia e Innovaciones Tecnologicas (IVIT); ArgentinaFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Prasad, B. V. Venkataram. Baylor College of Medicine. Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology; Estados UnidosFil: Prasad, B. V. Venkataram. Baylor College of Medicine. Department of Molecular Virology and Microbiology; Estados Unido

X-Pro peptides. A theoretical study of the hydrogen bonded conformations of (&#945;-aminoisobutyryl-L-prolyl)<SUB>n</SUB> sequences

Intramolecularly hydrogen bonded conformations of (Aib-Pro)n sequences have been analysed theoretically. Both 4&#8594;1 (C<SUB>10</SUB> and 3&#8594;1 (C<SUB>7</SUB> hydrogen bonded regular structures are shown to be stereochemically feasible. Conformational energies for the helical structures have been estimated using classical potential energy methods. Both C<SUB>10</SUB> and C<SUB>7</SUB> conformations have very similar energies. Pyrrolidine ring puckering has a pronounced effect on the energies, and only C&#947;-endo puckered Pro residues can be accommodated. The theoretical calculations using spectroscopic data suggest that the recently proposed novel 3<SUB>10</SUB> helical conformation for benzyloxycarbonyl(Aib-Pro)<SUB>4</SUB>-methyl ester is in solution, is indeed energetically and stereochemically favourable

Conformational analysis of small disulfide loops. Spectroscopic and theoretical studies on a synthetic cyclic tetrapeptide containing cystine

The conformational analysis of the synthetic peptide Boc-Cys-Pro-Val-Cys-NHMe has been carried out, as a model for small disulfide loops, in biologically active polypeptides. 'H NMR studies (270 MHz) establish that the Val(3) and Cys(4) NH groups are solvent shielded, while 13C studies establish an all-trans peptide backbone. Circular dichroism and Raman spectroscopy provide evidence for a right-handed twist of the disulfide bond. Analysis of the vicinal (JaB)c oupling constants for the two Cys residues establishes that XI - *60° for Cys(4), while some flexibility is suggested at Cys( 1). Conformational energy calculations, imposing intramolecular hydrogen bonding constraints, favor a P-turn (type I) structure with Pro(2)-Va1(3) as the corner residues. Theoretical and spectroscopic results are consistent with the presence of a transannular 4 - 1 hydrogen bond between Cys( 1) CO and Cys(4) NH groups, with the Val NH being sterically shielded from the solvent environment

Molecular structure of a cyclic tetrapeptide disulfide. A novel 3<SUB>10</SUB> helical conformation with an S-S bridge

The crystal structure of the cyclic peptide disulfide Boc-Cys-Pro-Aib-Cys-NHMe has been determined by X-ray diffraction. The peptide crystallizes in the space group P2<SUB>1</SUB>2<SUB>1</SUB>2<SUB>1</SUB>, with a = 8.646(1), b = 18.462(2), c = 19.678(3)&#197; and Z = 4. The molecules adopt a highly folded compact conformation, stabilized by two intramolecular 4 &#8594; 1 hydrogen bonds between the Cys (1) and Pro (2) CO groups and the Cys (4) and methylamide NH groups, respectively. The backbone conformational angles for the peptide lie very close to those expected for a 3<SUB>10</SUB> helix. The S-S bridge adopts a right handed twist with a dihedral angle of 82°. The structure illustrates the role of stereochemically constrained residues, in generating novel peptide conformations