Seeing the chemistry in biology with neutron crystallography

Langan, Paul; Chen, Julian C.-H.

doi:10.1039/c3cp51760h

Cited by 20 publications

(19 citation statements)

References 58 publications

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“…Owing to the need for the development of CA isoformspecific inhibitors against CA IX, and to gain a deeper understanding of its active-site architecture, our goal is to utilize joint neutron and X-ray structures of CA IX SV alone and in inhibitor complexes to fine-tune compounds to preferentially bind CA IX over CA II (Langan & Chen, 2013;Aggarwal et al, 2013;Kovalevsky et al, 2018). Previous neutron crystallographic studies of CA II in complex with clinically used inhibitors (for example brinzolamide, ethoxzolamide and acetazolamide) revealed the role and importance of water and hydrogen bonds in mediating ligandbinding interactions (Kovalevsky et al, 2018;Fisher et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Koruza

Lafumat

Nyblom

et al. 2019

Acta Crystallogr D Struct Biol

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Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX–inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

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Section: Introductionmentioning

confidence: 99%

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Koruza

Lafumat

Nyblom

et al. 2019

Acta Crystallogr D Struct Biol

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“…It is the only technique that can directly, and at high resolution, visualize H and its isotope deuterium, D. Unlike X-rays, neutrons are scattered very well by H/D atoms, similarly to the other common elements found in proteins, such as C, N, and O. This allows detailed analysis of water coordination, H bonding, ligand binding, and the protonation state of certain amino acids such as His and Lys (14,15). This information is crucial to understanding how H bonds and water mediate not only enzyme catalysis but also ligand binding.…”

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confidence: 99%

Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer

Michalczyk

Ünkefer

Schrader

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Human carbonic anhydrase II (HCA II) uses a Zn-bound OH − /H 2 O mechanism to catalyze the reversible hydration of CO 2 . This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pK a compared with free tyrosine. To further probe the pK a of this residue, NMR spectroscopic measurements of [ 13 C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pK a of Tyr7 and how pH and Tyr proximity to Zn affect hydrogenbonding interactions.proton transfer | neutron crystallography | perturbed pKa | solution NMR C arbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the interconversion of CO 2 and HCO 3 − . In humans, CAs are involved in a great variety of physiological processes, from respiration, ureagenesis, gluconeogenesis, and cerebrospinal fluid production to general acid/base homeostasis (1). The most abundant and well-characterized human CA isoform is human carbonic anhydrase II (HCA II), which displays a bellshaped pH dependence on catalytic activity, with a maximal k cat of 10 6 per second at pH 7.2. In the hydration direction, the first step of catalysis is a Zn-OH − -mediated nucleophilic attack on CO 2 to form HCO 3 − and one H + , leaving one water molecule bound to the zinc. The second, rate-limiting step occurs via proton transfer (PT) that removes one H + from the metal-bound water to regenerate the catalytic Zn-OH − (2). This PT step is thought to adopt classic Grotthuss-type hopping and it is mediated by a well-ordered network of hydrogen-bonded (H-bonded) water molecules that spans the ∼8-Å distance between the Znwater and proton-shuttling residue, His64 (Figs. 1-3). In previous neutron structures, the Zn-bound solvent has been observed to be an H 2 O (or exchanged D 2 O) and this may imply that H 2 O is the favored species over OH − in the absence of substrate (3). In chemical crystallography extended X-ray absorption fine structure can be used to determine Zn:Zn-solvent distances as a way to discriminate between H 2 O or OH − . For Zn-OH − the distance should be ∼1.80 Å and for Zn-H 2 O it should be closer to 2.0 Å (4). Despite the availability of ultra-high-resolution X-ray crystal structures of holo HCA II (no substrate present) the range of Zn:Zn-solvent distances observed has led to inconclusive results. Also, within the coordinate error it is unfeasible to determine the Zn-bound solvent species using distance with any certainty (5, 6).His64 is thought to be one component in the delivery of excess H + to the bulk solvent, resetting the a...

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“…Interested readers are referred to comprehensive reviews for historical, technical and scientific perspectives on neutron protein crystallography for further reading (Niimura & Podjarny, 2011;Blakeley, 2009;Blakeley et al, 2008Blakeley et al, , 2015Meilleur et al, 2006;Myles, 2006;O'Dell et al, 2016;Langan & Chen, 2013;Chen & Unkefer, 2017;Oksanen et al, 2017).…”

Section: Neutron Protein Crystallographymentioning

confidence: 99%

IMAGINE: neutrons reveal enzyme chemistry

Schröder

O’Dell

Myles

et al. 2018

Acta Cryst Sect D Struct Biol

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Neutron diffraction is exquisitely sensitive to the positions of H atoms in protein crystal structures. IMAGINE is a high-intensity, quasi-Laue neutron crystallography beamline developed at the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. This state-of-the-art facility for neutron diffraction has enabled detailed structural analysis of macromolecules. IMAGINE is especially suited to resolve individual H atoms in protein structures, enabling neutron protein structures to be determined at or near atomic resolutions from crystals with volumes of less than 1 mm and unit-cell edges of less than 150 Å. Beamline features include elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm focal spot at the sample position, and variable short- and long-wavelength cutoff optics that provide automated exchange between multiple wavelength configurations. This review gives an overview of the IMAGINE beamline at the HFIR, presents examples of the scientific questions being addressed at this beamline, and highlights important findings in enzyme chemistry that have been made using the neutron diffraction capabilities offered by IMAGINE.

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Seeing the chemistry in biology with neutron crystallography

Cited by 20 publications

References 58 publications

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX

Joint neutron crystallographic and NMR solution studies of Tyr residue ionization and hydrogen bonding: Implications for enzyme-mediated proton transfer

IMAGINE: neutrons reveal enzyme chemistry

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