Crystal structure of the ubiquitin‐like small archaeal modifier protein 2 from <i>Haloferax volcanii</i>

Li, Yunfeng; Maciejewski, Mark W.; Martin, Jonathan H.; Jin, Kai; Zhang, Yuhang; Maupin-Furlow, Julie A.; Hao, Bing

doi:10.1002/pro.2305

Cited by 8 publications

(9 citation statements)

References 51 publications

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“…D). The SAMP3 K62 modified by sampylation was somewhat analogous to Ub K48 in its location on 3D structure as determined recently by NMR . K62 was found at the carboxyl end of β3, which is part of the β‐grasp fold structure characteristic of Ub‐like proteins (Fig.…”

Section: Resultssupporting

confidence: 66%

Proteome targets of ubiquitin‐like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii

Dantuluri

Hepowit

et al. 2016

Proteomics

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Small archeal modifier proteins (SAMPs) are related to ubiquitin in tertiary structure and in their isopeptide linkage to substrate proteins. SAMPs also function in sulfur mobilization to form biomolecules such as molybdopterin and thiolated tRNA. While SAMP1 is essential for anaerobic growth and covalently attached to lysine residues of its molybdopterin synthase partner MoaE (K240 and K247), the full diversity of proteins modified by samp1ylation is not known. Here, we expand the knowledge of proteins isopeptide linked to SAMP1. LC-MS/MS analysis of -Gly-Gly signatures derived from SAMP1 S85R conjugates cleaved with trypsin was used to detect sites of sampylation (23 lysine residues) that mapped to 11 target proteins. Many of the identified target proteins were associated with sulfur metabolism and oxidative stress including MoaE, SAMP-activating E1 enzyme (UbaA), methionine sulfoxide reductase homologs (MsrA and MsrB), and the Fe-S assembly protein SufB. Several proteins were found to have multiple sites of samp1ylation, and the isopeptide linkage at SAMP3 lysines (K18, K55, and K62) revealed hetero-SAMP chain topologies. Follow-up affinity purification of selected protein targets (UbaA and MoaE) confirmed the LC-MS/MS results. 3D homology modeling suggested sampy1ylation is autoregulatory in inhibiting the activity of its protein partners (UbaA and MoaE), while occurring on the surface of some protein targets, such as SufB and MsrA/B. Overall, we provide evidence that SAMP1 is a ubiquitin-like protein modifier that is relatively specific in tagging its protein partners as well as proteins associated with oxidative stress response.

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Section: Resultssupporting

confidence: 66%

Proteome targets of ubiquitin‐like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii

Dantuluri

Hepowit

et al. 2016

Proteomics

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“…Here we extend our knowledge of post-translational mechanisms in archaea beyond SAMP1/2 conjugation by demonstrating that SAMP3 (recently shown by NMR structural studies to form a classic ␤-grasp fold in a salt-independent manner) (30) can act as a Ubl protein modifier in Hfx. volcanii.…”

supporting

confidence: 55%

Archaeal Ubiquitin-like SAMP3 is Isopeptide-linked to Proteins via a UbaA-dependent Mechanism

Miranda

Antelmann

Hepowit

et al. 2014

Molecular & Cellular Proteomics

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SAMP1 and SAMP2 are ubiquitin-like proteins that function as protein modifiers and are required for the production of sulfur-containing biomolecules in the archaeon Haloferax volcanii. Here we report a novel small archaeal modifier protein (named SAMP3) with a ␤-grasp fold and C-terminal diglycine motif characteristic of ubiquitin that is functional in protein conjugation in Hfx. volcanii. SAMP3 conjugates were dependent on the ubiquitin-activating E1 enzyme homolog of archaea (UbaA) for synthesis and were cleaved by the JAMM/MPN؉ domain metalloprotease HvJAMM1. Twenty-three proteins (28 lysine residues) were found to be isopeptide-linked to the C-terminal carboxylate of SAMP3, and 331 proteins were reproducibly found associated with SAMP3 in a UbaA-dependent manner based on tandem mass spectrometry (MS/MS) analysis. The molybdopterin (MPT) synthase large subunit homolog MoaE, found samp3ylated at conserved active site lysine residues in MS/MS analysis, was also shown to be covalently bound to SAMP3 by immunoprecipitation and tandem affinity purifications. HvJAMM1 was demonstrated to catalyze the cleavage of SAMP3 from MoaE, suggesting a mechanism of controlling MPT synthase activity. The levels of samp3ylated proteins and samp3 transcripts were found to be increased by the addition of dimethyl sulfoxide to aerobically growing cells. Thus, we propose a model in which samp3ylation is covalent and reversible and controls the activity of enzymes such as MPT synthase. Sampylation of MPT synthase may govern the levels of molybdenum cofactor available and thus facilitate the scavenging of oxygen prior to the transition to respiration with molybdenum-cofactor-containing terminal reductases that use alternative electron acceptors such as dimethyl sulfoxide. Overall, our study of SAMP3 provides new insight into the diversity of functional ubiquitin-like protein modifiers and the network of ubiquitin-like protein targets in Archaea. Molecular & Cellular

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“…(A) UbaA and SAMP1/3 were found to migrate as single protein bands and SAMP2 as two distinct protein bands, when separated by reducing SDS/PAGE and stained with Coomassie brilliant blue. The flexible β‐hinge region of SAMP2 was attributed to account for its unusual migration by SDS/PAGE, based on analysis of samples by electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF MS) that suggested protein homogeneity (Table S1). (B) Representative SEC elution profiles are presented which reveal UbaA and SAMP1 to be a homodimer (~ 55 kDa) and monomer (~ 10 kDa), respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Much like the bacterial MoaD binding to MoeB [28,32], the archaeal SAMP1 binds UbaA in the absence of ATP, with this association stabilized by ATP binding. By contrast, SAMP2, which has an extended C-terminal tail and unique bhinge region [27,33], binds UbaA like eukaryotic Ub binds E1 [34], through an ordered mechanism in which ATP binding precedes Ub/Ubl protein binding to the E1/E1-like enzyme. Interestingly, the downstream consequences of SAMP2 binding to UbaA are also eukaryotic-like (SAMP2 tags proteins for destruction by proteasomes, whereas SAMP1-modified proteins can be quite stable) [19,35].…”

Section: Ubaa Binding To Its Cognate Samp Is Influenced By Environmenmentioning

confidence: 99%

“…The binding of UbaA to SAMPs was assessed in vivo including an evaluation of the impact of culture conditions. To accomplish this, the UbaA and SAMPs were [27] was attributed to account for its unusual migration by SDS/PAGE, based on analysis of samples by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) that suggested protein homogeneity (Table S1). (B) Representative SEC elution profiles are presented which reveal UbaA and SAMP1 to be a homodimer (~55 kDa) and monomer (~10 kDa), respectively.…”

Section: Ubaa Associates With the Samps In Vivomentioning

confidence: 99%

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Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin‐like proteins of Archaea

et al. 2016

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Here we provide the first detailed biochemical study of a noncanonical E1-like enzyme with broad specificity for cognate ubiquitin-like (Ubl) proteins that mediates Ubl protein modification and sulfur mobilization to form molybdopterin and thiolated tRNA. Isothermal titration calorimetry and in vivo analyses proved useful in discovering that environmental conditions, ATP binding and Ubl type controlled the mechanism of association of the Ubl protein with its cognate E1-like enzyme (SAMP and UbaA of the archaeon Haloferax volcanii, respectively). Further analysis revealed ATP hydrolysis triggered the formation of thioester and peptide bonds within the Ubl:E1-like complex. Importantly, the thioester was an apparent precursor to Ubl protein modification but not sulfur mobilization. Comparative modeling to MoeB/ThiF guided the discovery of key residues within the adenylation domain of UbaA that were needed to bind ATP as well as residues that were specifically needed to catalyze the downstream reactions of sulfur mobilization and/or Ubl protein modification. UbaA was also found to be Ubl-automodified at lysine residues required for early (ATP binding) and late (sulfur mobilization) stages of enzyme activity revealing multiple layers of auto-regulation. Cysteine residues, distinct from the canonical E1 ‘active site’ cysteine, were found important in UbaA function supporting a model that this non-canonical E1 is structurally flexible in its active site to allow Ubl~adenylate, Ubl~E1-like thioester and cysteine persulfide(s) intermediates to form.

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Crystal structure of the ubiquitin‐like small archaeal modifier protein 2 from Haloferax volcanii

Cited by 8 publications

References 51 publications

Proteome targets of ubiquitin‐like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii

Proteome targets of ubiquitin‐like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii

Archaeal Ubiquitin-like SAMP3 is Isopeptide-linked to Proteins via a UbaA-dependent Mechanism

Mechanistic insight into protein modification and sulfur mobilization activities of noncanonical E1 and associated ubiquitin‐like proteins of Archaea

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