STRUCTURAL BIOMEDICINE: CHARACTERIZATION OF THE STRUCTURAL BASIS IN PROTEIN-DRUG RECOGNITION IN DIFFERENT HUMAN DISEASES

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

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Ancient and conserved functional interplay between Bcl-2 family proteins in the mitochondrial pathway of apoptosis

Science Advances ◽

10.1126/sciadv.abc4149 ◽

2020 ◽

Vol 6 (40) ◽

pp. eabc4149 ◽

Cited By ~ 2

Author(s):

Nikolay Popgeorgiev ◽

Jaison D Sa ◽

Lea Jabbour ◽

Suresh Banjara ◽

Trang Thi Minh Nguyen ◽

...

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Cancer Cells ◽

Mitochondrial Pathway ◽

Structural Basis ◽

Mitochondrial Outer Membrane ◽

Chemotherapy Treatment ◽

Trichoplax Adhaerens ◽

Binding Groove

In metazoans, Bcl-2 family proteins are major regulators of mitochondrially mediated apoptosis; however, their evolution remains poorly understood. Here, we describe the molecular characterization of the four members of the Bcl-2 family in the most primitive metazoan, Trichoplax adhaerens. All four trBcl-2 homologs are multimotif Bcl-2 group, with trBcl-2L1 and trBcl-2L2 being highly divergent antiapoptotic Bcl-2 members, whereas trBcl-2L3 and trBcl-2L4 are homologs of proapoptotic Bax and Bak, respectively. trBax expression permeabilizes the mitochondrial outer membrane, while trBak operates as a BH3-only sensitizer repressing antiapoptotic activities of trBcl-2L1 and trBcl-2L2. The crystal structure of a trBcl-2L2:trBak BH3 complex reveals that trBcl-2L2 uses the canonical Bcl-2 ligand binding groove to sequester trBak BH3, indicating that the structural basis for apoptosis control is conserved from T. adhaerens to mammals. Finally, we demonstrate that both trBax and trBak BH3 peptides bind selectively to human Bcl-2 homologs to sensitize cancer cells to chemotherapy treatment.

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Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate

Scientific Reports ◽

10.1038/s41598-019-53301-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Jolanta Krucinska ◽

Michael N. Lombardo ◽

Heidi Erlandsen ◽

Akram Hazeen ◽

Searle S. Duay ◽

...

Keyword(s):

Bacterial Infections ◽

Antibacterial Properties ◽

High Energy ◽

Structural Basis ◽

Gram Negative Bacteria ◽

E Coli ◽

Promising Target ◽

Antibiotic Discovery ◽

Growth And Reproduction

AbstractMany years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

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Towards Understanding the Structural Basis of Endogenous Transcription in dsRNA Viruses

Microscopy and Microanalysis ◽

10.1017/s1431927600007339 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 89-90

Author(s):

B. V. Venkataram Prasad ◽

J. A. Lawton ◽

C. Q. -Y. Zeng ◽

M. K. Estes

Keyword(s):

Structural Basis ◽

Structural Studies ◽

Rotavirus A ◽

Internal Organization ◽

Computer Image Processing ◽

Structural Details ◽

Intact Core ◽

Icosahedral Viruses ◽

Dsrna Viruses

In double-stranded RNA (dsRNA) viruses found in animals, bacteria, and yeast, the genome is transcribed within the structurally intact core of the virion with extraordinary efficiency. How is the genome transcribed within the confines of the intact core particles? How are the nascent mRNA transcripts released through the capsid layers? Critical to our understanding of the structural basis of endogenous transcription is the characterization of the internal organization in terms of genome and transcription enzymes in these viruses. Our recent structural studies on rotavirus, a prototypical dsRNA virus, have begun to provide a better understanding of this process.Rotaviruses are the major pathogens of infantile gastroenteritis. These are complex and relatively large (1000 Å in diameter), nonenveloped icosahedral viruses. The structure of rotavirus is composed of three concentric capsid protein layers enclosing eleven strands of dsRNA (Fig. 1a). Although previous structural studies using electron cryomicroscopy and computer image processing, have described the overall architecture of the rotavirus, the structural details of internal organization has been elusive.

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Genetic and structural characterization of an amino acid dimorphism in glycoprotein Ib alpha involved in platelet transfusion refractoriness

Blood ◽

10.1182/blood.v79.11.3086.3086 ◽

1992 ◽

Vol 79 (11) ◽

pp. 3086-3090 ◽

Cited By ~ 58

Author(s):

M Murata ◽

K Furihata ◽

F Ishida ◽

SR Russell ◽

J Ware ◽

...

Keyword(s):

Platelet Transfusion ◽

Platelet Reactivity ◽

Recombinant Expression ◽

Structural Basis ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

Definitive Evidence ◽

Naturally Occurring ◽

Alpha Gene

Abstract The platelet-specific alloantigen, Siba, located within the alpha- subunit of the glycoprotein (GP) Ib-IX membrane receptor, has been found to be involved in the pathogenesis of platelet transfusion refractoriness. We have identified the existence of a naturally occurring threonine/methionine dimorphism at position 145 of the GPIb alpha sequence, and determined that the Siba antigen corresponds to the molecule containing methionine145. The diallelic codons can be detected by restriction enzyme analysis of amplified genomic DNA fragments from the GPIb alpha gene. Evaluation of 61 healthy blood donors showed that the allele frequencies are 89% and 11% for the threonine145 and methionine145 codons, respectively. A positive correlation exists between platelet reactivity with the anti-Siba antibody and the presence of a methionine145-encoding allele. Moreover, recombinant expression of two soluble GPIb alpha fragments differing only at residue 145, provided definitive evidence that the human anti-Siba antibody reacts only with the molecule containing methionine145. These results explain the structural basis of the Siba human alloantigen system and define screening methodologies useful in transfusion medicine to match donor and recipient platelets accordingly.

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