Small-angle X-ray scattering studies of enzymes

Byer, Amanda S.; Pei, Xiaokun; Patterson, Michael G.; Ando, Nozomi

doi:10.1016/j.cbpa.2022.102232

Cited by 19 publications

(10 citation statements)

References 43 publications

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“…We therefore began by comprehensively characterizing this enzyme using SAXS. SAXS is unique in its ability to yield direct structural information on an entire solution conformational ensemble and, when applied carefully, can detect subtle conformational changes in flexible, multi-domain proteins 28,29 . To determine how the cobalamin oxidation state correlates with conformation, we compared three representative states of MetH: the CH 3 -Cob(III) and Cob(I) states from the turnover cycle and the inactive, His-on Cob(II) state from the reactivation cycle (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy

Watkins

Wang

Burnim

et al. 2023

Preprint

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Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH3-H4folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling ofS-adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies onEscherichia coliMetH have shown that this flexible, multi-domain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a "divide and conquer" approach. In this study, we investigateE. coliMetH and a thermophilic homolog fromThermus filiformisusing small-angle X-ray scattering (SAXS), single-particle cryo-electron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present the first structural description of MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH3-H4folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of theT. filiformisMetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.

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Section: Resultsmentioning

confidence: 99%

Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy

Watkins

Wang

Burnim

et al. 2023

Preprint

Self Cite

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“…Although their static structures can be determined by x-ray crystallography ( Blow and Steitz, 1970 ) or cryo-EM ( Tsai et al , 2022 ), their functions are difficult to observe directly with atomic precision ( Johnson, 2013 ; Schmidt, 2020 ; and Wilson, 2022 ). Methods with time-resolution on enzyme solutions such as absorption spectroscopy in combination with stop-flow mixing experiments ( Johnson, 2013 ) or time-resolved Small and Wide Angle x-ray Scattering (SAXS/WAXS) ( Kim et al , 2012 ; Bjorling et al , 2015 ; Grant, 2018 ; and Byer et al , 2022 ) provide an avenue to investigate enzyme catalysis. However, they all lack atomic resolution.…”

Section: Enzymesmentioning

confidence: 99%

Biological function investigated by time-resolved structure determination

Schmidt

2023

Structural Dynamics

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Inspired by recent progress in time-resolved x-ray crystallography and the adoption of time-resolution by cryo-electronmicroscopy, this article enumerates several approaches developed to become bigger/smaller, faster, and better to gain new insight into the molecular mechanisms of life. This is illustrated by examples where chemical and physical stimuli spawn biological responses on various length and time-scales, from fractions of Ångströms to micro-meters and from femtoseconds to hours.

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“…At 77 kDa, TG2 is a relatively small protein for cryo-EM, and the highly dynamic nature of its conformational changes provides an additional complication. X-ray crystallography has been used to obtain 3D structures for TG2; although the constraints imposed by formation of a crystal lattice may mask the conformational and oligomeric transitions that TG2 undergoes in response to different ligands and inhibitors (33, 37–39, 41, 42, 58). This prompted us to utilize small angle X-ray scattering (SAXS), as this method is performed in solution and at room temperature making it well suited to study the conformational dynamics of flexible proteins and to distinguish between the open and closed forms of TG2 (59–61).…”

Section: Introductionmentioning

confidence: 99%

Defining the conformational states that enable transglutaminase 2 to promote cancer cell survival versus cell death

Aplin,

Zielinski,

Pabit

et al. 2024

Preprint

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Transglutaminase 2 (TG2) is a GTP-binding/protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that maintain the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotide-bound TG2 adopts a monomeric closed conformation while calcium-bound TG2 assumes an open conformational state that can form higher order oligomers. SAXS analysis also suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time-resolved SAXS to show that LM11 increases the ability of calcium to drive TG2 to an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.Significance StatementThe multi-functional protein transglutaminase 2 (TG2) undergoes large conformational changes in response to nucleotide and calcium binding, resulting in diverse cellular effects that can differentially promote either cancer cell survival or cell death. Previous biochemical and structural characterizations have revealed that TG2 primarily adopts two conformational states, a closed nucleotide-bound conformation, and an open calcium-bound conformation. In this study, we use advanced structural methods to describe the conformational changes associated with TG2 activation and inhibition and define the mechanism by which small molecule inhibitors maintain TG2 in a structural state that kill cancer cells.

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Small-angle X-ray scattering studies of enzymes

Cited by 19 publications

References 43 publications

Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy

Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryo-electron microscopy

Biological function investigated by time-resolved structure determination

Defining the conformational states that enable transglutaminase 2 to promote cancer cell survival versus cell death

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