Structural insight into activity enhancement and inhibition of H64A carbonic anhydrase II by imidazoles

Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2and HCO3−, respectively. The reaction follows a ping-pong mechanism, in which the rate-limiting step is the transfer of a proton from the zinc-bound solvent (OH−/H2O) in/out of the active siteviaHis64, which is widely believed to be the proton-shuttling residue. The decreased catalytic activity (∼20-fold lower with respect to the wild type) of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines, to almost the wild-type level. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives (imidazole, 1-methylimidazole, 2-methylimidazole and 4-methylimidazole) have been determined and reveal multiple binding sites. Two of these imidazole binding sites have been identified that mimic the positions of the `in' and `out' rotamers of His64 in wild-type CA II, while another directly inhibits catalysis by displacing the zinc-bound solvent. The data presented here not only corroborate the importance of the imidazole side chain of His64 in proton transfer during CA catalysis, but also provide a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used at higher concentrations) the activity of H64A CA II.

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Insights into Activity Enhancement of H64A Carbonic Anhydrase by Imidazoles

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314091967 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C803-C803

Author(s):

Mayank Aggarwal ◽

Chingkuang Tu ◽

David Silverman ◽

Robert McKenna

Keyword(s):

Binding Sites ◽

Ph Regulation ◽

Side Chain ◽

Carbonic Anhydrases ◽

Physiological Processes ◽

Multiple Binding Sites ◽

Zinc Metalloenzymes ◽

Number Of Binding Sites ◽

Activity Enhancement ◽

Proton Shuttling

Human carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the hydration and dehydration of CO2 and HCO3-, respectively. The reaction follows a ping-pong mechanism, where the rate limiting step is the transfer of a proton from the zinc-bound solvent out of the active site, via His64 which is widely believed to be the proton shuttling residue. Being involved in a number of physiological processes such as respiration, pH regulation, ureagenesis etc., CAs are therapeutic targets for inhibition to treat various diseases. However, the physiologically dominant isoform is CA II, which is catalytically highly efficient and is easily crystallizable. Thus, most of our knowledge in the design of CA inhibitors with pharmacological applications is based on detailed CA II crystallographic studies. The catalytic activity of a variant of CA II in which His64 is replaced with Ala (H64A CA II) can be enhanced by exogenous proton donors/acceptors, usually derivatives of imidazoles and pyridines. This article examines the mechanism through which this activity enhancement might occur. X-ray crystal structures of H64A CA II in complex with four imidazole derivatives have been determined and reveal multiple binding sites. We have identified two molecules of imidazoles that bind in region that is otherwise occupied by the "in" and "out" dual conformation of the side chain of His64 in wild-type CA II. The data presented here not only corroborates the importance of imidazole side chain of His64 in proton transfer during CA catalysis, but also provides a complete structural understanding of the mechanism by which imidazoles enhance (and inhibit when used in higher concentrations) the activity of H64A CA II. In addition to inhibition of CA by these imidazoles, the presence of a large number of binding sites also gives insights and preliminary data required to fragment addition approach of drug design against CA.

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Interactions of Brf1 Peptides with the Tetratricopeptide Repeat-Containing Subunit of TFIIIC Inhibit and Promote Preinitiation Complex Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.00689-06 ◽

2006 ◽

Vol 26 (16) ◽

pp. 5946-5956 ◽

Cited By ~ 11

Author(s):

Yanling Liao ◽

Robyn D. Moir ◽

Ian M. Willis

Keyword(s):

Binding Sites ◽

Structural Model ◽

Rna Polymerase Iii ◽

Directed Assembly ◽

Initiation Factor ◽

Tetratricopeptide Repeat ◽

Rate Limiting Step ◽

Multiple Binding Sites ◽

Starting Point ◽

Multiple Binding

ABSTRACT The binding of Brf1 to the tetratricopeptide repeat (TPR)-containing transcription factor IIIC (TFIIIC) subunit (Tfc4) represents a rate-limiting step in the ordered assembly of the RNA polymerase III initiation factor TFIIIB. Tfc4 contains multiple binding sites for Brf1 within its amino terminus and adjacent TPR arrays, but the access of Brf1 to these sites is limited by autoinhibition. Moreover, the Brf1 binding sites in Tfc4 overlap with sites important for the subsequent recruitment of another TFIIIB subunit, Bdp1, implying that repositioning of Brf1 is required after its initial interaction with Tfc4. As a starting point for dissecting the steps in TFIIIC-directed assembly of TFIIIB, we conducted yeast two-hybrid screens of Brf1 peptide libraries against different TPR-containing Tfc4 fragments. Short, biochemically active peptides were identified in three distinct regions of Brf1. Two peptides defined conserved but distal regions of Brf1 that participate in stable binding of Brf1 to TFIIIC-DNA. Remarkably, a third peptide that binds specifically to TPR6-9 of Tfc4 was found to promote the formation of both TFIIIC-DNA and Brf1-TFIIIC-DNA complexes and to reduce the mobility of these complexes in native gels. The data are consistent with this peptide causing a conformational change in TFIIIC that overcomes Tfc4 autoinhibition of Brf1 binding and suggest a structural model for the Brf1-Tfc4 interaction.

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Protein Determinants of Insertional Specificity for the Drosophila Gypsy Retrovirus

Genetics ◽

10.1093/genetics/158.3.1101 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1101-1110

Author(s):

Mariano Labrador ◽

Victor G Corces

Keyword(s):

Binding Sites ◽

Germ Line ◽

Wild Type ◽

In Ovo ◽

Multiple Binding Sites ◽

Genomic Location ◽

Multiple Binding ◽

Insertion Sites ◽

Dna Fragment ◽

Late Stages

Abstract The gypsy retrovirus invades the germ line of Drosophila females, inserting with a high frequency into the ovo locus. Gypsy insertion sites in ovo are clustered within a region in the promoter of the ovo gene that contains multiple binding sites for the OvoA and OvoB proteins. We found that a 1.3-kb DNA fragment containing this region is able to confer gypsy insertional specificity independent of its genomic location. The frequency of gypsy insertions into the ovo gene is significantly lower in wild-type females than in ovoD1 females. In addition, gypsy insertions in ovoD1 females occur during most stages of germ-line development whereas insertions in wild-type females occur only in late stages. This pattern of temporally specific insertions, as well as the higher frequency of insertion in ovoD1 females, correlates with the presence of the OvoA or OvoD1 proteins. The results suggest that gypsy insertional specificity might be determined by the binding of the OvoA repressor isoform to the promoter region of the gene.

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Faculty Opinions recommendation of Multiple binding sites for the general anesthetic isoflurane identified in the nicotinic acetylcholine receptor transmembrane domain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.5804956.5786054 ◽

2010 ◽

Author(s):

Edward Bertaccini

Keyword(s):

Acetylcholine Receptor ◽

Nicotinic Acetylcholine Receptor ◽

Binding Sites ◽

Transmembrane Domain ◽

General Anesthetic ◽

Nicotinic Acetylcholine ◽

Multiple Binding Sites ◽

Multiple Binding

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NMDAR PAMs: Multiple Chemotypes for Multiple Binding Sites

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666191011095341 ◽

2019 ◽

Vol 19 (24) ◽

pp. 2239-2253 ◽

Cited By ~ 2

Author(s):

Paul J. Goldsmith

Keyword(s):

Binding Sites ◽

Receptor Activation ◽

Research Area ◽

Allosteric Modulators ◽

Nonselective Cation Channels ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Excitatory Neurotransmission ◽

Psychiatric Conditions ◽

High Level

The N-methyl-D-aspartate receptor (NMDAR) is a member of the ionotropic glutamate receptor (iGluR) family that plays a crucial role in brain signalling and development. NMDARs are nonselective cation channels that are involved with the propagation of excitatory neurotransmission signals with important effects on synaptic plasticity. NMDARs are functionally and structurally complex receptors, they exist as a family of subtypes each with its own unique pharmacological properties. Their implication in a variety of neurological and psychiatric conditions means they have been a focus of research for many decades. Disruption of NMDAR-related signalling is known to adversely affect higherorder cognitive functions (e.g. learning and memory) and the search for molecules that can recover (or even enhance) receptor output is a current strategy for CNS drug discovery. A number of positive allosteric modulators (PAMs) that specifically attempt to overcome NMDAR hypofunction have been discovered. They include various chemotypes that have been found to bind to several different binding sites within the receptor. The heterogeneity of chemotype, binding site and NMDAR subtype provide a broad landscape of ongoing opportunities to uncover new features of NMDAR pharmacology. Research on NMDARs continues to provide novel mechanistic insights into receptor activation and this review will provide a high-level overview of the research area and discuss the various chemical classes of PAMs discovered so far.

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Effects of Multiple Binding Sites on Studies of Hydrogen Bonding between Nitroxide Radicals and Solvent Molecules

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19930047 ◽

1993 ◽

Vol 58 (1) ◽

pp. 47-52 ◽

Cited By ~ 2

Author(s):

Imad Al-Bala'a ◽

Richard D. Bates

Keyword(s):

Hydrogen Bonding ◽

Magnetic Resonance ◽

Binding Site ◽

Binding Sites ◽

Hyperfine Coupling Constant ◽

Hydrogen Donor ◽

Complex Formation Constants ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Solvent Molecules

The role of more than one binding site on a nitroxide free radical in magnetic resonance determinations of the properties of the complex formed with a hydrogen donor is examined. The expression that relates observed hyperfine couplings in EPR spectra to complex formation constants and concentrations of each species in solution becomes much more complex when multiple binding sites are present, but reduces to a simpler form when binding at the two sites occurs independently and the binding at the non-nitroxide site does not produce significant differences in the hyperfine coupling constant in the complexed radical. Effects on studies of hydrogen bonding between multiple binding site nitroxides and hydrogen donor solvent molecules by other magnetic resonance methods are potentially more extreme.

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