skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Neutron structure of human carbonic anhydrase II in complex with methazolamide: Mapping the solvent and hydrogen-bonding patterns of an effective clinical drug

Abstract

Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the interconversion of CO 2 and HCO 3 , and their inhibitors have long been used as diuretics and as a therapeutic treatment for many disorders such as glaucoma and epilepsy. Acetazolamide (AZM) and methazolamide (MZM, a methyl derivative of AZM) are two of the classical CA inhibitory drugs that have been used clinically for decades. The jointly refined X-ray/neutron structure of MZM in complex with human CA isoform II (hCA II) has been determined to a resolution of 2.2 Å with an R cryst of ~16.0%. Presented in this article, along with only the second neutron structure of a clinical drug-bound hCA, is an in-depth structural comparison and analyses of differences in hydrogen-bonding network, water-molecule orientation and solvent displacement that take place upon the binding of AZM and MZM in the active site of hCA II. Even though MZM is slightly more hydrophobic and displaces more waters than AZM, the overall binding affinity ( K i) for both of the drugs against hCA II is similar (~10 n M). The plausible reasons behind this finding have also been discussed using molecular dynamics and X-ray crystal structures of hCA II–MZM determined at cryotemperature andmore » room temperature. Furthermore, this study not only allows a direct comparison of the hydrogen bonding, protonation states and solvent orientation/displacement of AZM and MZM, but also shows the significant effect that the methyl derivative has on the solvent organization in the hCA II active site.« less

Authors:
 [1];  [1];  [2];  [3];  [2];  [4]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  3. European Spallation Source, Lund (Sweden)
  4. Univ. of Florida, Gainesville, FL (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1326115
Grant/Contract Number:
AC05-00OR22725
Resource Type:
Journal Article: Published Article
Journal Name:
IUCrJ
Additional Journal Information:
Journal Volume: 3; Journal Issue: 5; Journal ID: ISSN 2052-2525
Publisher:
International Union of Crystallography
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; human carbonic anhydrase; acetazolamide; methazolamide; neutron structure; drug binding

Citation Formats

Aggarwal, Mayank, Kovalevsky, Andrey Y., Velazquez, Hector, Fisher, S. Zoe, Smith, Jeremy C., and McKenna, Robert. Neutron structure of human carbonic anhydrase II in complex with methazolamide: Mapping the solvent and hydrogen-bonding patterns of an effective clinical drug. United States: N. p., 2016. Web. doi:10.1107/S2052252516010514.
Aggarwal, Mayank, Kovalevsky, Andrey Y., Velazquez, Hector, Fisher, S. Zoe, Smith, Jeremy C., & McKenna, Robert. Neutron structure of human carbonic anhydrase II in complex with methazolamide: Mapping the solvent and hydrogen-bonding patterns of an effective clinical drug. United States. doi:10.1107/S2052252516010514.
Aggarwal, Mayank, Kovalevsky, Andrey Y., Velazquez, Hector, Fisher, S. Zoe, Smith, Jeremy C., and McKenna, Robert. 2016. "Neutron structure of human carbonic anhydrase II in complex with methazolamide: Mapping the solvent and hydrogen-bonding patterns of an effective clinical drug". United States. doi:10.1107/S2052252516010514.
@article{osti_1326115,
title = {Neutron structure of human carbonic anhydrase II in complex with methazolamide: Mapping the solvent and hydrogen-bonding patterns of an effective clinical drug},
author = {Aggarwal, Mayank and Kovalevsky, Andrey Y. and Velazquez, Hector and Fisher, S. Zoe and Smith, Jeremy C. and McKenna, Robert},
abstractNote = {Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the interconversion of CO2 and HCO3–, and their inhibitors have long been used as diuretics and as a therapeutic treatment for many disorders such as glaucoma and epilepsy. Acetazolamide (AZM) and methazolamide (MZM, a methyl derivative of AZM) are two of the classical CA inhibitory drugs that have been used clinically for decades. The jointly refined X-ray/neutron structure of MZM in complex with human CA isoform II (hCA II) has been determined to a resolution of 2.2 Å with an Rcryst of ~16.0%. Presented in this article, along with only the second neutron structure of a clinical drug-bound hCA, is an in-depth structural comparison and analyses of differences in hydrogen-bonding network, water-molecule orientation and solvent displacement that take place upon the binding of AZM and MZM in the active site of hCA II. Even though MZM is slightly more hydrophobic and displaces more waters than AZM, the overall binding affinity (Ki) for both of the drugs against hCA II is similar (~10 nM). The plausible reasons behind this finding have also been discussed using molecular dynamics and X-ray crystal structures of hCA II–MZM determined at cryotemperature and room temperature. Furthermore, this study not only allows a direct comparison of the hydrogen bonding, protonation states and solvent orientation/displacement of AZM and MZM, but also shows the significant effect that the methyl derivative has on the solvent organization in the hCA II active site.},
doi = {10.1107/S2052252516010514},
journal = {IUCrJ},
number = 5,
volume = 3,
place = {United States},
year = 2016,
month = 7
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1107/S2052252516010514

Citation Metrics:
Cited by: 2works
Citation information provided by
Web of Science

Save / Share:
  • Carbonic anhydrases (CAs; EC 4.2.1.1) catalyze the interconversion of CO 2 and HCO 3 , and their inhibitors have long been used as diuretics and as a therapeutic treatment for many disorders such as glaucoma and epilepsy. Acetazolamide (AZM) and methazolamide (MZM, a methyl derivative of AZM) are two of the classical CA inhibitory drugs that have been used clinically for decades. The jointly refined X-ray/neutron structure of MZM in complex with human CA isoform II (hCA II) has been determined to a resolution of 2.2 Å with an R cryst of ~16.0%. Presented in this article, along with onlymore » the second neutron structure of a clinical drug-bound hCA, is an in-depth structural comparison and analyses of differences in hydrogen-bonding network, water-molecule orientation and solvent displacement that take place upon the binding of AZM and MZM in the active site of hCA II. Even though MZM is slightly more hydrophobic and displaces more waters than AZM, the overall binding affinity ( K i) for both of the drugs against hCA II is similar (~10 n M). The plausible reasons behind this finding have also been discussed using molecular dynamics and X-ray crystal structures of hCA II–MZM determined at cryotemperature and room temperature. Furthermore, this study not only allows a direct comparison of the hydrogen bonding, protonation states and solvent orientation/displacement of AZM and MZM, but also shows the significant effect that the methyl derivative has on the solvent organization in the hCA II active site.« less
  • The neutron structure of wild type human carbonic anhydrase II at pH 7.8 has been determined to 2.0 resolution. Detailed analysis and comparison to the previous determined structure at pH 10.0 shows important differences in protonation of key catalytic residues in the active site as well as a rearrangement of the hydrogen bonded water network. For the first time, a completed hydrogen bonded network stretching from the Zn-bound solvent to the proton shuttling residue His64 has been directed observed.
  • Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) and its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Protein X-ray crystallography has seen a progressive shift from data collection at cool/room temperature (277–298 K) to data collection at cryotemperature (100 K) because of its ease of crystal preparation and the lessening of the detrimental effects of radiation-induced crystal damage, with 20–25%(v/v) glycerol (GOL) being the preferred choice of cryoprotectant. Here, a case study of the effects of cryoprotectants on the kinetics of carbonic anhydrase II (CA II) andmore » its inhibition by the clinically used inhibitor acetazolamide (AZM) is presented. Comparative studies of crystal structure, kinetics, inhibition and thermostability were performed on CA II and its complex with AZM in the presence of either GOL or sucrose. These results suggest that even though the cryoprotectant GOL was previously shown to be directly bound in the active site and to interact with AZM, it affects neither the thermostability of CA II nor the binding of AZM in the crystal structure or in solution. However, addition of GOL does affect the kinetics of CA II, presumably as it displaces the water proton-transfer network in the active site.« less
  • A full-length cDNA clone encoding human mitochondrial carbonic anhydrase (CA), CA V, was isolated from a human liver cDNA library. The 1123-bp cDNA includes a 55-bp 5[prime] untranslated region, a 915-bp open reading frame, and a 153-bp 3[prime] untranslated region. Expression of the cDNA in COS cells produced active enzyme. The 34-kDa precursor and 30-kDa mature form of CA V were identified on Western blots of COS-cell homogenates by a CA V-specific antibody raised to a synthetic peptide corresponding to the C-terminal 17 aa of CA V. Both 34-kDa and 30-kDa bands were also present in mitochondria isolated from transfectedmore » COS cells, whereas only the 30-kDa band was present in mitochondria isolate from normal human liver. The N-terminal sequence determined directly on the 30-kDa soluble CA purified from transfected COS cells indicated that processing of the precursor to mature human CA V involves removal of a 38-aa mitochondrial leader sequence. The 267-aa sequence deduced for mature human CA V shows 30-49% similarity to amino acid sequences of previously characterized human CAs (CA I-CA VII) and 76% similarity to the corresponding amino acid sequence deduced from the mouse cDNA. PCR analysis of DNAs from human-rodent somatic cell hybrids localized the gene for CA V to human chromosome 16, the same chromosome to which CA VII has previously been mapped. 48 refs., 4 figs., 1 tab.« less
  • No abstract prepared.