Crystal Structure of Wild-Type Tryptophan Synthase Complexed with the Natural Substrate Indole-3-glycerol Phosphate†

ABSTRACT The role of receptor recognition in the emergence of virulent viruses was investigated in the infection of severe combined immunodeficient (SCID) mice by the apathogenic prototype strain of the parvovirus minute virus of mice (MVMp). Genetic analysis of isolated MVMp viral clones (n = 48) emerging in mice, including lethal variants, showed only one of three single changes (V325M, I362S, or K368R) in the common sequence of the two capsid proteins. As was found for the parental isolates, the constructed recombinant viruses harboring the I362S or the K368R single substitutions in the capsid sequence, or mutations at both sites, showed a large-plaque phenotype and lower avidity than the wild type for cells in the cytotoxic interaction with two permissive fibroblast cell lines in vitro and caused a lethal disease in SCID mice when inoculated by the natural oronasal route. Significantly, the productive adsorption of MVMp variants carrying any of the three mutations selected through parallel evolution in mice showed higher sensitivity to the treatment of cells by neuraminidase than that of the wild type, indicating a lower affinity of the viral particle for the sialic acid component of the receptor. Consistent with this, the X-ray crystal structure of the MVMp capsids soaked with sialic acid (N-acetyl neuraminic acid) showed the sugar allocated in the depression at the twofold axis of symmetry (termed the dimple), immediately adjacent to residues I362 and K368, which are located on the wall of the dimple, and approximately 22 Å away from V325 in a threefold-related monomer. This is the first reported crystal structure identifying an infectious receptor attachment site on a parvovirus capsid. We conclude that the affinity of the interactions of sialic-acid-containing receptors with residues at or surrounding the dimple can evolutionarily regulate parvovirus pathogenicity and adaptation to new hosts.

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Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

Biochemical Journal ◽

10.1042/bj3590065 ◽

2001 ◽

Vol 359 (1) ◽

pp. 65-75 ◽

Cited By ~ 25

Author(s):

Valeria MENCHISE ◽

Catherine CORBIER ◽

Claude DIDIERJEAN ◽

Michele SAVIANO ◽

Ettore BENEDETTI ◽

...

Keyword(s):

Crystal Structure ◽

Proton Transfer ◽

Chlamydomonas Reinhardtii ◽

Catalytic Mechanism ◽

Structural Data ◽

Careful Analysis ◽

Wild Type ◽

Thioredoxin H ◽

Dynamics Simulations

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

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Generation of an Isogenic Collection of Yeast Actin Mutants and Identification of Three Interrelated Phenotypes

Genetics ◽

10.1093/genetics/157.2.533 ◽

2001 ◽

Vol 157 (2) ◽

pp. 533-543

Author(s):

Johanna L Whitacre ◽

Dana A Davis ◽

Kurt A Toenjes ◽

Sharon M Brower ◽

Alison E M Adams

Keyword(s):

Crystal Structure ◽

Growth Rates ◽

Small Region ◽

Wild Type ◽

Large Collection ◽

Growth Defects ◽

Cytoskeletal Function ◽

Highly Correlated ◽

The Relationship ◽

Mutant Cells

Abstract A large collection of yeast actin mutations has been previously isolated and used in numerous studies of actin cytoskeletal function. However, the various mutations have been in congenic, rather than isogenic, backgrounds, making it difficult to compare the subtle phenotypes that are characteristic of these mutants. We have therefore placed 27 mutations in an isogenic background. We used a subset of these mutants to compare the degree to which different actin alleles are defective in sporulation, endocytosis, and growth on NaCl-containing media. We found that the three phenotypes are highly correlated. The correlations are specific and not merely a reflection of general growth defects, because the phenotypes are not correlated with growth rates under normal conditions. Significantly, those actin mutants exhibiting the most severe phenotypes in all three processes have altered residues that cluster to a small region of the actin crystal structure previously defined as the fimbrin (Sac6p)-binding site. We examined the relationship between endocytosis and growth on salt and found that shifting wild-type or actin mutant cells to high salt reduces the rate of α-factor internalization. These results suggest that actin mutants may be unable to grow on salt because of additive endocytic defects (due to mutation and salt).

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An alkaline phosphatase mutant of Pseudomonas aeruginosa. 1. Effects of regulatory, structural, and environmental shifts on enzyme function

Canadian Journal of Microbiology ◽

10.1139/m78-070 ◽

1978 ◽

Vol 24 (4) ◽

pp. 427-432 ◽

Cited By ~ 1

Author(s):

M. L. Marceau-Day ◽

D. F Day ◽

J. M. Ingram

Keyword(s):

Pseudomonas Aeruginosa ◽

Alkaline Phosphatase ◽

Core Region ◽

Significant Activity ◽

Wild Type ◽

Glycerol Phosphate ◽

The Core ◽

Nitrophenyl Phosphate ◽

Mutant Cells

An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both β-glycerol phosphate (βGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of βGP versus pNPP, whereas this ratio was reversed to 1:9 βGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide(LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.

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The dissociation mechanism of processive cellulases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913398116 ◽

2019 ◽

Vol 116 (46) ◽

pp. 23061-23067 ◽

Cited By ~ 10

Author(s):

Josh V. Vermaas ◽

Riin Kont ◽

Gregg T. Beckham ◽

Michael F. Crowley ◽

Mikael Gudmundsson ◽

...

Keyword(s):

Crystal Structure ◽

Molecular Mechanisms ◽

Soluble Sugars ◽

Dissociation Rate ◽

Replica Exchange ◽

Wild Type ◽

Replica Exchange Molecular Dynamics ◽

Hamiltonian Replica Exchange ◽

Biochemical Measurements ◽

Rate Limiting

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

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Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

Journal of Virology ◽

10.1128/jvi.02044-17 ◽

2018 ◽

Vol 92 (7) ◽

Cited By ~ 9

Author(s):

Xiyuan Wu ◽

Zixuan Liu ◽

Xiaohui Ding ◽

Danwei Yu ◽

Huamian Wei ◽

...

Keyword(s):

Crystal Structure ◽

Cross Resistance ◽

Fusion Inhibitor ◽

Viral Fusion ◽

Wild Type ◽

Genetic Pathway ◽

Fusion Inhibitors ◽

Helical Peptide ◽

Mimic Peptide ◽

Hiv 1

ABSTRACTSC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors.IMPORTANCET20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

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The crystal structure of the tryptophan synthase β2 subunit from the hyperthermophile Pyrococcus furiosus

European Journal of Biochemistry ◽

10.1111/j.1432-1033.2004.04191.x ◽

2004 ◽

Vol 271 (13) ◽

pp. 2624-2635 ◽

Cited By ~ 11

Author(s):

Yusaku Hioki ◽

Kyoko Ogasahara ◽

Soo Jae Lee ◽

Jichun Ma ◽

Masami Ishida ◽

...

Keyword(s):

Crystal Structure ◽

Pyrococcus Furiosus ◽

Tryptophan Synthase

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Co-Crystal Structures of 7-Azaindole Inhibitors of Wild-Type and T315I Imatinib-Resistant Mutant Forms of the BCR-ABL Tyrosine Kinase.

Blood ◽

10.1182/blood.v110.11.1018.1018 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1018-1018

Author(s):

Hal A. Lewis ◽

Fred Zhang ◽

Richard Romero ◽

Pierre-Yves Bounaud ◽

Mark E. Wilson ◽

...

Keyword(s):

Crystal Structure ◽

Tyrosine Kinase ◽

Crystal Structures ◽

Kinase Domain ◽

Hinge Region ◽

Wild Type ◽

X Ray ◽

Abl Kinase ◽

Imatinib Resistant ◽

Mutant Forms

Abstract Chronic myelogenous leukemia (CML) arises from uncontrolled cell growth driven by a constitutively active BCR-ABL fusion protein tyrosine kinase, which is the product of the pathognomonic Philadelphia chromosomal translocation. Imatinib mesylate (Gleevec) is a BCR-ABL inhibitor used as a first line treatment of CML. Although imatinib is highly effective in chronic phase CML, in advanced disease patients frequently relapse due to the emergence of drug resistance. Approximately two-thirds of resistance is caused by point mutations in the BCR-ABL kinase domain, which give rise to active mutant forms of the enzyme that are insensitive to Gleevec. The T315I mutation represents one of the most common causes of resistance, is resistant to the second generation BCR-ABL inhibitors dasatinib and nilotinib, and represents an important and challenging target for discovery of next generation targeted CML treatments. We have applied X-ray crystallographic screening of our FAST™ fragment library and structure-guided hit-to-lead optimization to identify potent inhibitors of both wild-type and T315I mutant BCR-ABL. These efforts yielded a 7-azaindole compound series that exhibits binding to and inhibition of both wild-type and T315I BCR-ABL. Methods: Wild-type (with Y393F) and T315I Abl kinase domain protein were expressed in E. coli and purified to homogeneity. These proteins were crystallized in the presence of a reference inhibitor followed by addition of the 7-azaindole series compounds soaked into the preformed crystals to displace the reference compound, giving the desired co-crystal. X-ray diffraction data were recorded at the company’s proprietary synchrotron beamline SGX-CAT at the Advanced Photon Source. Three-dimensional enzyme-inhibitor co-crystal structures were determined by molecular replacement and refined to permit modeling of bound ligand. Results: Both wild-type and T315I Abl structures revealed enzyme in the active conformation with inhibitors bound to the kinase hinge region. The crystal structure of 2-amino-5-[3-(1-ethyl-1H-pyrazol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl]-N,N-dimethylbenzamide in complex with T315I, illustrates the typical binding mode which is independent of the 315 residue, and therefore accounts for the compound inhibiting the T315I mutant form of BCR-ABL (see figure). The inhibitor binds to the hinge region of ABL utilizing hydrogen bonding to backbone carbonyl of Glu316 and NH of Met318, with the pyrazole ring stacking in a lipophilic pocket between Phe382 and Tyr253. In addition, the benzamide carbonyl participates in a hydrogen bond interactioin with the backbone-NH of Glu249 of the p-loop. Conclusions: X-ray crystallographic fragment screening and co-crystal structure studies have been successfully employed in discovery/optimization of 7-azaindole series compounds, yielding potent, selective inhibitors of both wild-type and imatinib-resistant forms of BCR-ABL. Figure Figure

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