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Subunit Association (subunit + association)

Distribution by Scientific Domains

Chemistry	80%

Selected Abstracts

Probing nonnucleoside inhibitor-induced active-site distortion in HIV-1 reverse transcriptase by transient kinetic analyses

PROTEIN SCIENCE, Issue 8 2007
Qing Xia
Abstract Nonnucleoside reverse transcriptase inhibitors (NNRTI) are a group of structurally diverse compounds that bind to a single site in HIV-1 reverse transcriptase (RT), termed the NNRTI-binding pocket (NNRTI-BP). NNRTI binding to RT induces conformational changes in the enzyme that affect key elements of the polymerase active site and also the association between the two protein subunits. To determine which conformational changes contribute to the mechanism of inhibition of HIV-1 reverse transcription, we used transient kinetic analyses to probe the catalytic events that occur directly at the enzyme's polymerase active site when the NNRTI-BP was occupied by nevirapine, efavirenz, or delavirdine. Our results demonstrate that all NNRTI,RT,template/primer (NNRTI,RT,T/P) complexes displayed a metal-dependent increase in dNTP binding affinity (Kd) and a metal-independent decrease in the maximum rate of dNTP incorporation (kpol). The magnitude of the decrease in kpol was dependent on the NNRTI used in the assay: Efavirenz caused the largest decrease followed by delavirdine and then nevirapine. Analyses that were designed to probe direct effects on phosphodiester bond formation suggested that the NNRTI mediate their effects on the chemistry step of the DNA polymerization reaction via an indirect manner. Because each of the NNRTI analyzed in this study exerted largely similar phenotypic effects on single nucleotide addition reactions, whereas each of them are known to exert differential effects on RT dimerization, we conclude that the NNRTI effects on subunit association do not directly contribute to the kinetic mechanism of inhibition of DNA polymerization. [source]

Structural asymmetry and intersubunit communication in muscle creatine kinase

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2007
Jeffrey F. Ohren
The structure of a transition-state analog complex of a highly soluble mutant (R134K) of rabbit muscle creatine kinase (rmCK) has been determined to 1.65,Å resolution in order to elucidate the structural changes that are required to support and regulate catalysis. Significant structural asymmetry is seen within the functional homodimer of rmCK, with one monomer found in a closed conformation with the active site occupied by the transition-state analog components creatine, MgADP and nitrate. The other monomer has the two loops that control access to the active site in an open conformation and only MgADP is bound. The N-terminal region of each monomer makes a substantial contribution to the dimer interface; however, the conformation of this region is dramatically different in each subunit. Based on this structural evidence, two mutational modifications of rmCK were conducted in order to better understand the role of the amino-terminus in controlling creatine kinase activity. The deletion of the first 15 residues of rmCK and a single point mutant (P20G) both disrupt subunit cohesion, causing the dissociation of the functional homodimer into monomers with reduced catalytic activity. This study provides support for a structural role for the amino-terminus in subunit association and a mechanistic role in active-site communication and catalytic regulation. [source]

Limited proteolysis analysis of the ribosome is affected by subunit association

BIOPOLYMERS, Issue 6 2009
Daisy-Malloy Hamburg
Abstract Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from misfolding leading to their eventual degradation within the cell, is limited because of the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X-ray crystallography, and cryo-EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits. Isolated subunits were analyzed using limited proteolysis and MALDI-MS and the results were compared with previous data obtained from 70S ribosomes. Generally, ribosomal proteins were found to be more stable in 70S ribosomes than in their isolated subunits, consistent with a reduction in conformational flexibility on subunit assembly. This approach demonstrates that limited proteolysis combined with MALDI-MS can reveal structural changes to ribosomes on subunit assembly or disassembly, and provides the appropriate benchmark data from 30S, 50S, and 70S proteins to enable studies of ribosome assembly intermediates. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 410,422, 2009. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source]

Molecular Diversity Of Vascular Potassium Channel Isoforms

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2002
Victoria P Korovkina
SUMMARY 1. One essential role for potassium channels in vascular smooth muscle is to buffer cell excitation and counteract vasoconstrictive influences. Several molecular mechanisms regulate potassium channel function. The interaction of these mechanisms may be one method for fine-tuning potassium channel activity in response to various physiological and pathological challenges. 2. The most prevalent K+ channels in vascular smooth muscle are large-conductance calcium- and voltage-sensitive channels (maxi-K channels) and voltage-gated channels (Kv channels). Both channel types are complex molecular structures consisting of a pore-forming , -subunit and an ancillary , -subunit. The maxi-K and Kv channel , -subunits assemble as tetramers and have S4 transmembrane domains that represent the putative voltage sensor. While most vascular smooth muscle cells identified to date contain both maxi-K and Kv channels, the expression of individual , -subunit isoforms and , -subunit association occurs in a tissue-specific manner, thereby providing functional specificity. 3. The maxi-K channel , -subunit derives its molecular diversity by alternative splicing of a single-gene transcript to yield multiple isoforms that differ in their sensitivity to intracellular Ca2+ and voltage, cell surface expression and post- translational modification. The ability of this channel to assemble as a homo- or heterotetramer allows for fine-tuning control to intracellular regulators. Another level of diversity for this channel is in its association with accessory , -subunits. Multiple , -subunits have been identified that can arise either from separate genes or alternative splicing of a , -subunit gene. The maxi-K channel , -subunits modulate the channel's Ca2+ and voltage sensitivity and kinetic and pharmacological properties. 4. The Kv channel , -subunit derives its diverse nature by the expression of several genes. Similar to the maxi-K channel, this channel has been shown to assemble as a homo- and heterotetramer, which can significantly change the Kv current phenotype in a given cell type. Association with a number of the ancillary , -subunits affects Kv channel function in several ways. Beta-subunits can induce inactivating properties and act as chaperones, thereby regulating channel cell-surface expression and current kinetics. [source]

Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase complexed with 6-phosphogluconate

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2009
Scott Cameron
Two crystal structures of recombinant Geobacillus stearothermophilus 6-phosphogluconate dehydrogenase (Gs6PDH) in complex with the substrate 6-phosphogluconate have been determined at medium resolution. Gs6PDH shares significant sequence identity and structural similarity with the enzymes from Lactococcus lactis, sheep liver and the protozoan parasite Trypanosoma brucei, for which a range of structures have previously been reported. Comparisons indicate that amino-acid sequence conservation is more pronounced in the two domains that contribute to the architecture of the active site, namely the N-terminal and C-terminal domains, compared with the central domain, which is primarily involved in the subunit,subunit associations required to form a stable dimer. The active-site residues are highly conserved, as are the interactions with the 6-phosphogluconate. There is interest in 6PDH as a potential drug target for the protozoan parasite T. brucei, the pathogen responsible for African sleeping sickness. The recombinant T. brucei enzyme has proven to be recalcitrant to enzyme,ligand studies and a surrogate protein might offer new opportunities to investigate and characterize 6PDH inhibitors. The high degree of structural similarity, efficient level of expression and straightforward crystallization conditions mean that Gs6PDH may prove to be an appropriate model system for structure-based inhibitor design targeting the enzyme from Trypanosoma species. [source]