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

Koruza, Katarina; Lafumat, Bénédicte; Nyblom, Maria; Mahon, Brian P.; Knecht, Wolfgang; McKenna, Robert; Fisher, S.Z.

doi:10.1107/s2059798319010027

Cited by 14 publications

(6 citation statements)

References 41 publications

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“…A superposition of the final room-temperature D UOX-8AZA-W1 model with that of the 1.75 Å resolution UOX-8AZA-W1 X-ray structure solved at 4 C (277 K) (PDB entry 1r51; Retailleau et al, 2004) or with that of H/D-exchanged 1.1 Å resolution H/D UOX-8AZA-Cl À X-ray structure solved at 100 K (PDB entry 4n9v; Oksanen et al, 2014) reveals essentially no differences, with RMSD values of 0.18 and 0.22 Å , respectively, for all common main-chain atoms. Thus, in line with what has been reported for other proteins (Koruza et al, 2019), partial or complete deuterium-labelling does not induce major structural changes in UOX.…”

Section: Perdeuterated a Flavus Uox ( D Uox) For Neutron Crystallogrsupporting

confidence: 91%

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis

McGregor

Földes

Bui

et al. 2021

Int Union Crystallogr J

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Cofactor-independent urate oxidase (UOX) is an ∼137 kDa tetrameric enzyme essential for uric acid (UA) catabolism in many organisms. UA is first oxidized by O2 to dehydroisourate (DHU) via a peroxo intermediate. DHU then undergoes hydration to 5-hydroxyisourate (5HIU). At different stages of the reaction both catalytic O2 and water occupy the `peroxo hole' above the organic substrate. Here, high-resolution neutron/X-ray crystallographic analysis at room temperature has been integrated with molecular dynamics simulations to investigate the hydration step of the reaction. The joint neutron/X-ray structure of perdeuterated Aspergillus flavus UOX in complex with its 8-azaxanthine (8AZA) inhibitor shows that the catalytic water molecule (W1) is present in the peroxo hole as neutral H2O, oriented at 45° with respect to the ligand. It is stabilized by Thr57 and Asn254 on different UOX protomers as well as by an O—H...π interaction with 8AZA. The active site Lys10–Thr57 dyad features a charged Lys10–NH3 + side chain engaged in a strong hydrogen bond with Thr57OG1, while the Thr57OG1–HG1 bond is rotationally dynamic and oriented toward the π system of the ligand, on average. Our analysis offers support for a mechanism in which W1 performs a nucleophilic attack on DHUC5 with Thr57HG1 central to a Lys10-assisted proton-relay system. Room-temperature crystallography and simulations also reveal conformational heterogeneity for Asn254 that modulates W1 stability in the peroxo hole. This is proposed to be an active mechanism to facilitate W1/O2 exchange during catalysis.

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Section: Perdeuterated a Flavus Uox ( D Uox) For Neutron Crystallogrsupporting

confidence: 91%

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis

McGregor

Földes

Bui

et al. 2021

Int Union Crystallogr J

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“…3(a)]. The conserved tertiary and secondary structures indicate that perdeuteration did not have a significant impact on the overall protein fold, as has been demonstrated in many neutron crystallographic studies of other proteins (Artero et al, 2005;Haupt et al, 2014;Langan et al, 2014;Cuypers, Mason et al, 2013;Yee et al, 2019;Liu et al, 2007;Koruza et al, 2019). The r.m.s.d.…”

Section: Secondary and Tertiary Structures Are Retainedmentioning

confidence: 69%

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

Ramos

Laux

Haertlein

et al. 2021

Int Union Crystallogr J

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This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in Escherichia coli followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast Pichia pastoris, as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated Gallus gallus protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97–Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.

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“…One of the common ways of testing this isomorphism is through a comparison of the X-ray analyses of analogous structures, and there is now a steadily growing database of these comparisons. For crystalline systems, the isomorphism usually holds good to high resolution (Gamble et al, 1994;Cooper et al, 1998;Meilleur et al, 2004;Artero et al, 2005;Liu et al, 2007;Fisher & Helliwell, 2008;Cuypers et al, 2013;Yee et al, 2016Yee et al, , 2019Koruza et al, 2019;Ramos et al, 2021). For the HEWL work described here, this is, to our knowledge, the first time that a detailed comparative study has been made of hydrogenated and perdeuterated analogs of a refolded protein.…”

Section: Introductionmentioning

confidence: 88%

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme

Ramos¹,

Laux²,

Haertlein³

et al. 2021

Acta Crystallogr D Struct Biol

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The biological function of a protein is intimately related to its structure and dynamics, which in turn are determined by the way in which it has been folded. In vitro refolding is commonly used for the recovery of recombinant proteins that are expressed in the form of inclusion bodies and is of central interest in terms of the folding pathways that occur in vivo. Here, biophysical data are reported for in vitro-refolded hydrogenated hen egg-white lysozyme, in combination with atomic resolution X-ray diffraction analyses, which allowed detailed comparisons with native hydrogenated and refolded perdeuterated lysozyme. Distinct folding modes are observed for the hydrogenated and perdeuterated refolded variants, which are determined by conformational changes to the backbone structure of the Lys97–Gly104 flexible loop. Surprisingly, the structure of the refolded perdeuterated protein is closer to that of native lysozyme than that of the refolded hydrogenated protein. These structural differences suggest that the observed decreases in thermal stability and enzymatic activity in the refolded perdeuterated and hydrogenated proteins are consequences of the macromolecular deuteration effect and of distinct folding dynamics, respectively. These results are discussed in the context of both in vitro and in vivo folding, as well as of lysozyme amyloidogenesis.

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

Cited by 14 publications

References 41 publications

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis

Joint neutron/X-ray crystal structure of a mechanistically relevant complex of perdeuterated urate oxidase and simulations provide insight into the hydration step of catalysis

Structural insights into protein folding, stability and activity using in vivo perdeuteration of hen egg-white lysozyme

The impact of folding modes and deuteration on the atomic resolution structure of hen egg-white lysozyme

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