The protein data bank: A computer-based archival file for macromolecular structures

Bernstein, F.C.; Koetzle, Thomas F.; Williams, G. J. B.; Meyer, Edgar F.; Brice, Michael D.; Rodgers, John R.; Kennard, Olga; Shimanouchi, Takehiko; Tasumi, Mitsuo

doi:10.1016/s0022-2836(77)80200-3

Cited by 8,186 publications

(2,675 citation statements)

References 8 publications

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“…Crystals diffracted routinely below 1.2 Å and were subjected to synchrotron data collection. Results were compared with the four GacA (RmlD) homologues that have been deposited in the Protein Data Bank (PDB) (Bernstein et al ., 1977) (Supp. Fig.…”

Section: Resultsmentioning

confidence: 99%

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

Beek

Breton

Ferenbach

et al. 2015

Molecular Microbiology

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SummaryThe sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A S treptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium S almonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gac A in a S. mutans rml D knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gac A as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.

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Section: Resultsmentioning

confidence: 99%

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

Beek

Breton

Ferenbach

et al. 2015

Molecular Microbiology

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“…Starting coordinates [16,21,22] were taken from the Protein Data Bank [23] according to the space group (Table 1). Conventional crystallographic refinement (rigid body, positional and temperature factor) was carried out using XPLOR [24,25].…”

Section: Methodsmentioning

confidence: 99%

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

1995

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Crystal structures of DX9065a and a related bisamidino-aryl inhibitor specific for the blood-clotting factor Xa have been solved in complex with bovine ~-trypsin to a resolution of 1.9 A. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibiturs. Few direct contacts (predominantly in the S1 pocke0 are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three-site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobie surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific '60-insertion loop', thus conferring selectivity against thrombin.

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“…The structure refinements of the T state models were initiated by extracting the atomic data (Ifrp) from the Protein Data Bank (Bernstein et al, 1977). The structure used was a model of the T state refined at 2 A resolution to an R-factor of 18.3% (Xue et al, 1994) (Fig.…”

Section: Quality and Description Of The Structuresmentioning

confidence: 99%

Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli

et al. 1996

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The active site of pig kidney fructose-l,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-l,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-l,6-bisphosphate was reduced IO-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chern 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243 + Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.

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The protein data bank: A computer-based archival file for macromolecular structures

Cited by 8,186 publications

References 8 publications

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

Crystal structures of the active site mutant (Arg‐243 → Ala) in the T and R allosteric states of pig kidney fructose‐1,6‐bisphosphatase expressed in escherichia coli

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