Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the <i>Clostridium acetobutylicum</i> Spore Photoproduct Lyase

Yang, Linlin; Adhikari, Jagat; Gross, Michael L.; Li, Lei

doi:10.1111/php.12697

Cited by 3 publications

(6 citation statements)

References 40 publications

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“…The collated peptide list from multiple MS2 runs and the MS1 raw files from nondeuterated runs were analyzed using HDX Workbench (Omics Informatics, Honolulu, HI) (49). The centroid masses of isotopic envelopes and deuterium levels were determined as described previously (50, 51).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus X Protein Function Requires Zinc Binding

Ramakrishnan

Xing

Beran

et al. 2019

J Virol

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The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function. IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

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Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus X Protein Function Requires Zinc Binding

Ramakrishnan

Xing

Beran

et al. 2019

J Virol

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“…As shown in the previous studies, TpT-

{SO}_{2}^{-}

was generated as the major product from the SP TpT repair by the C141A mutant in which the intrinsic H-donor to the thymine allylic radical, cysteine 141, is removed (Chandor-Proust et al, 2008; Yang et al, 2012). Additionally, TpT-

{SO}_{2}^{-}

was generated at ~20% yield (together with 80% TpT) during the repair of dinucleotide SP TpT by the SPL from the bacterium Clostridium acetobutylicum ( Ca ) (Yang et al, 2017). Different from the SPL ( Bs ) in which the conserved cysteine (C141) and tyrosine (Y99) are located on the same side of the enzyme binding pocket, the conserved cysteine (C74) in SPL ( Ca ) is projected to be at the opposite side to the conserved tyrosine, as indicated by the structural studies (Benjdia et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…Such a requirement determines that SPL ( Ca ) possesses a very flexible substrate binding pocket. As a consequence, C74 ( Ca ) is less likely to be well-positioned as the H-donor, especially when dinucleotide SP TpT, the smallest substrate, is used (Yang et al, 2017). This subsequently suggests that the thymine allylic radical can readily dissociate from the enzyme and be quenched by the externally added sodium dithionite.…”

Section: Discussionmentioning

confidence: 99%

“…Iron content was determined using o -bathophenanthroline (OBP) as described by Fish (1988) and sulfide assays were carried out using the method described by Beinert (1983). The detailed assay description can be found in our previous publications (Yang et al, 2011, 2012, 2017). …”

Section: Methodsmentioning

confidence: 99%

“…As revealed in previous studies (Ciccimaro and Blair, 2010; Liu et al, 2014; Wagner et al, 2015), linear responses between the intensity of mass spec signals and the amount of the analytes were observed, demonstrating the feasibility of using this LC-MRM-MS assay for quantitative analysis. The HPLC experiments used a ZORBAX Eclipse plus C18 column 4.6 × 50 mm (3.5 μm in particle size, Agilent Technologies, Santa Clara, CA) at ambient temperature, following the experimental procedures described in our previous studies (Yang et al, 2017). …”

Section: Methodsmentioning

confidence: 99%

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Insights into the Activity Change of Spore Photoproduct Lyase Induced by Mutations at a Peripheral Glycine Residue

Yang

2017

Front. Chem.

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UV radiation triggers the formation of 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP), in the genomic DNA of bacterial endospores. These SPs, if not repaired in time, may lead to genome instability and cell death. SP is mainly repaired by spore photoproduct lyase (SPL) during spore outgrowth via an unprecedented protein-harbored radical transfer pathway that is composed of at least a cysteine and two tyrosine residues. This mechanism is consistent with the recently solved SPL structure that shows all three residues are located in proximity and thus able to participate in the radical transfer process during the enzyme catalysis. In contrast, an earlier in vivo mutational study identified a glycine to arginine mutation at the position 168 on the B. subtilis SPL that is >15 Å away from the enzyme active site. This mutation appears to abolish the enzyme activity because endospores carrying this mutant were sensitive to UV light. To understand the molecular basis for this rendered enzyme activity, we constructed two SPL mutations G168A and G168R, examined their repair of dinucleotide SP TpT, and found that both mutants exhibit reduced enzyme activity. Comparing with the wildtype (WT) SPL enzyme, the G168A mutant slows down the SP TpT repair by 3~4-fold while the G168R mutant by ~ 80-fold. Both mutants exhibit a smaller apparent (DV) kinetic isotope effect (KIE) but a bigger competitive (DV/K) KIE than that by the WT SPL. Moreover, the G168R mutant also produces a large portion of the abortive repair product TpT-SO2−; the formation of which indicates that cysteine 141 is no longer well positioned as the H-donor to the thymine allylic radical intermediate. All these data imply that the mutation at the remote glycine 168 residue alters the enzyme 3D structure, subsequently reducing the SPL activity by changing the positions of the essential amino acids involved in the radical transfer process.

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Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

Zhu,

Iavarone,

Klinman

2024

ACS Cent. Sci.

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Interactions among proteins and peptides are essential for many biological activities including the tailoring of peptide substrates to produce natural products. The first step in the production of the bacterial redox cofactor pyrroloquinoline quinone (PQQ) from its peptide precursor is catalyzed by a radical SAM (rSAM) enzyme, PqqE. We describe the use of hydrogen−deuterium exchange mass spectrometry (HDX-MS) to characterize the structure and conformational dynamics in the protein−protein and protein− peptide complexes necessary for PqqE function. HDX-MS-identified hotspots can be discerned in binary and ternary complex structures composed of the peptide PqqA, the peptide-binding chaperone PqqD, and PqqE. Structural conclusions are supported by sizeexclusion chromatography coupled to small-angle X-ray scattering (SEC-SAXS). HDX-MS further identifies reciprocal changes upon the binding of substrate peptide and S-adenosylmethionine (SAM) to the PqqE/PqqD complex: long-range conformational alterations have been detected upon the formation of a quaternary complex composed of PqqA/PqqD/PqqE and SAM, spanning nearly 40 Å, from the PqqA binding site in PqqD to the PqqE active site Fe 4 S 4 . Interactions among the various regions are concluded to arise from both direct contact and distal communication. The described experimental approach can be readily applied to the investigation of protein conformational communication among a large family of peptide-modifying rSAM enzymes.

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Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the Clostridium acetobutylicum Spore Photoproduct Lyase

Cited by 3 publications

References 40 publications

Hepatitis B Virus X Protein Function Requires Zinc Binding

Hepatitis B Virus X Protein Function Requires Zinc Binding

Insights into the Activity Change of Spore Photoproduct Lyase Induced by Mutations at a Peripheral Glycine Residue

Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Local and Long-Distance Interactions within the Multicomponent Radical SAM Enzyme, PqqE

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