Small Archaeal Modifier Protein Research Articles

Evolutionary diversification of the autophagy-related ubiquitin-like conjugation systems

ABSTRACT Two autophagy-related (ATG) ubiquitin-like conjugation systems, the ATG12 and ATG8 systems, play important roles in macroautophagy. While multiple duplications and losses of the ATG conjugation system proteins are found in different lineages, the extent to which the underlying systems diversified across eukaryotes is not fully understood. Here, in order to understand the evolution of the ATG conjugation systems, we constructed a transcriptome database consisting of 94 eukaryotic species covering major eukaryotic clades and systematically identified ATG conjugation system components. Both ATG10 and the C-terminal glycine of ATG12 are essential for the canonical ubiquitin-like conjugation of ATG12 and ATG5. However, loss of ATG10 or the C-terminal glycine of ATG12 occurred at least 16 times in a wide range of lineages, suggesting that possible covalent-to-non-covalent transition is not limited to the species that we previously reported such as Alveolata and some yeast species. Some species have only the ATG8 system (with conjugation enzymes) or only ATG8 (without conjugation enzymes). More than 10 species have ATG8 homologs without the conserved C-terminal glycine, and Tetrahymena has an ATG8 homolog with a predicted transmembrane domain, which may be able to anchor to the membrane independent of the ATG conjugation systems. We discuss the possibility that the ancestor of the ATG12 and ATG8 systems is more similar to ATG8. Overall, our study offers a whole picture of the evolution and diversity of the ATG conjugation systems among eukaryotes, and provides evidence that functional diversifications of the systems are more common than previously thought. Abbreviations: APEAR: ATG8–PE association region; ATG: autophagy-related; LIR: LC3-interacting region; NEDD8: neural precursor cell expressed, developmentally down-regulated gene 8; PE: phosphatidylethanolamine; SAMP: small archaeal modifier protein; SAR: Stramenopiles, Alveolata, and Rhizaria; SMC: structural maintenance of chromosomes; SUMO: small ubiquitin like modifier; TACK: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota; UBA: ubiquitin like modifier activating enzyme; UFM: ubiquitin fold modifier; URM: ubiquitin related modifier.

Effects of ionic strength on the folding and stability of SAMP1, a ubiquitin-like halophilic protein

Our knowledge of the folding behavior of proteins from extremophiles is limited at this time. These proteins may more closely resemble the primordial proteins selected in early evolution under extreme conditions. The small archaeal modifier protein 1 (SAMP1) studied in this report is an 87-residue protein with a β-grasp fold found in the halophile Haloferax volcanii from the Dead Sea. To gain insight into the effects of salt on the stability and folding mechanism of SAMP1, we conducted equilibrium and kinetic folding experiments as a function of sodium chloride concentration. The results revealed that increasing ionic strength accelerates refolding and slows down unfolding of SAMP1, giving rise to a pronounced salt-induced stabilization. With increasing NaCl concentration, the rate of folding observed via a combination of continuous-flow (0.1–2 ms time range) and stopped-flow measurements (>2 ms) exhibited a >100-fold increase between 0.1 and 1.5 M NaCl and leveled off at higher concentrations. Using the Linderström-Lang smeared charge formalism to model electrostatic interactions in ground and transition states encountered during folding, we showed that the observed salt dependence is dominated by Debye-Hückel screening of electrostatic repulsion among numerous negatively charged residues. Comparisons are also drawn with three well-studied mesophilic members of the β-grasp superfamily: protein G, protein L, and ubiquitin. Interestingly, the folding rate of SAMP1 in 3 M sodium chloride is comparable to that of protein G, ubiquitin, and protein L at lower ionic strength. The results indicate the important role of electrostatic interactions in protein folding and imply that proteins have evolved to minimize unfavorable charge-charge interactions under their specific native conditions.

The nature of persistent interactions in two model β-grasp proteins reveals the advantage of symmetry in stability.

Two proteins within the β-grasp superfamily, the B1-domain of protein G and the small archaeal modifier protein 1, were investigated to elucidate the key determinants of structural stability at the level of individual interactions. These symmetrical proteins both contain two β-hairpins which form a sheet flanked by a central α-helix. They were subjected to high temperature molecular dynamics simulations and the detailed behavior of each long-range interaction was characterized. The results revealed that in GB1 the most stable region was the C-terminal hairpin and in SAMP1 it was the opposite, the N-terminal hairpin. Experimental results for GB1 support this finding. In conclusion, it appears that the difference in the location and number of hydrophobic interactions dictate the differential stability which is accommodated due to structural symmetry of the β-grasp fold. Thus, the hairpins are interchangeable and in nature this lends itself to adaptability and flexibility.

The ubiquitin-like modification by ThiS and ThiF in Escherichia coli

Escherichia coli, one of the most well-studied gram-negative bacterial species, encodes two ubiquitin-like proteins (UBLs), ThiS and MoaD. The studies on prokaryotic UBLs such as Pup, and small archaeal modifier protein have revealed the function of UBLs. However, in gram-negative bacteria, the functions of UBLs in protein modification are still poorly understood to date. Here, we report that ThiS, which has a β-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, is able to form protein conjugates in vivo and in vitro. We also constructed in vitro ThiS conjugation (thisylation) system and identified the modified lysine sites by MS/MS, this provides an essential platform for studying the UBLs thisylation system in E. coli. The modification system is dependent on lysine 83 (ATPase activity site) and cysteine 169 (zinc binding site) in ThiF and three important substrates, GroEL, PriC, FtsA, were found to be covalently modified by this system in vitro. Taken together, this study provided evidence that the protein conjugation function of β-grasp fold UBLs is conserved in the three major evolutionary lineages of life.

Haloferax volcanii UbaA, catalytic engine for sampylation and sulfur transfer

The Small Archaeal Modifier Proteins (SAMPs) from Haloferax volcanii belong to the group of ubiquitin like proteins (Ubls) that act both as protein modifiers and sulfur carriers. The E1-like enzyme UbaA is essential for SAMP activation and therefore required for both sampylation and sulfur transfer. Here, we provide a commentary on the thorough characterization of UbaA by J. Maupin-Furlow and colleagues.

Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea.

ABSTRACTThe molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself was not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN+ metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis.

Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners

A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895.

Prokaryotic ubiquitin-like protein modification.

Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers.

Ubiquitin‐like protein modification and proteasomes in Archaea (98.1)

Archaea encode a streamlined version of the eukaryotic ubiquitin‐proteasome system. In archaea, 20S proteasomes associate with AAA+ ATPases of simple subunit composition in the energy‐dependent degradation of proteins. More recently, we demonstrate that ubiquitin‐fold proteins of archaea (named small archaeal modifier proteins or SAMPs) are linked by isopeptide bonds to protein targets. This archaeal mechanism of attachment (named sampylation) appears less complex than ubiquitylation with a single ubiquitin‐activating E1 homolog (UbaA) functioning in the absence of ubiquitin‐conjugating E2 or ubiquitin‐ligase E3 enzymes. An archaeal isopeptidase composed of a single JAMM (JAB1/MPN/Mov34 metalloenzyme) domain is found to remove SAMPs from diverse protein conjugates, providing evidence that sampylation is reversible and likely regulated. Sites of sampylation, mapped to 36 lysines of 28 proteins by high‐throughput MS/MS analysis, were modeled in relationship to 3D‐structure and used to guide in vivo analysis and provide a basis for elucidating the biological function of sampylation. Based on this work, we propose that sampylation targets proteins for proteasome‐mediated proteolysis, modifies the catalytic active site of enzymes, modulates DNA‐binding protein interactions and regulates protein‐protein interactions.

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

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 β-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.

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

The discovery of ubiquitin-like small archaeal modifier protein 2 (SAMP2) that forms covalent polymeric chains in Haloferax volcanii has generated tremendous interest in the function and regulation of this protein. At present, it remains unclear whether the Hfx. volcanii modifier protein SAMP1 has such polyubiquitinating-like activity. Although SAMP1 and SAMP2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. To better understand the mechanism of SAMP2 conjugation, we have sought to characterize the biophysical and structural properties of the protein from Hfx. volcanii. SAMP2 is only partially structured under mesohalic solution conditions and adopts a well-folded compact conformation in the presence of 2.5M of NaCl. Its 2.3-Å-resolution crystal structure reveals a characteristic α/β central core domain and a unique β-hinge motif. This motif anchors an unusual C-terminal extension comprising the diglycine tail as well as two lysine residues that can potentially serve to interlink SAMP2 moieties. Mutational alternation of the structural malleability of this β-hinge motif essentially abolishes the conjugation activity of SAMP2 in vivo. In addition, NMR structural studies of the putative ubiquitin-like protein HVO_2177 from Hfx. volcanii show that like SAMP1, HVO_2177 forms a classic β-grasp fold in a salt-independent manner. These results provide insights into the structure-function relationship of sampylating proteins of fundamental importance in post-translational protein modification and environmental cues in Archaea.

Ionic strength‐dependent conformations of a ubiquitin‐like small archaeal modifier protein (SAMP1) fromHaloferax volcanii

Eukaryotic ubiquitin and ubiquitin-like systems play crucial roles in various cellular biological processes. In this work, we determined the solution structure of SAMP1 from Haloferax volcanii by NMR spectroscopy. Under low ionic conditions, SAMP1 presented two distinct conformations, one folded β-grasp and the other disordered. Interestingly, SAMP1 underwent a conformational conversion from disorder to order with ion concentration increasing, indicating that the ordered conformation is the functional form of SAMP1 under the physiological condition of H. volcanii. Furthermore, SAMP1 could interact with proteasome-activating nucleotidase B, supposing a potential role of SAMP1 in the protein degradation pathway mediated by proteasome.

Ionic strength-dependent conformations of a ubiquitin-like small archaeal modifier protein (SAMP2) from Haloferax volcanii

Ubiquitin-like proteins play important roles in diverse biological processes. In this study, we present an unexpected finding that a ubiquitin-like small archaeal modifier protein (SAMP2) from Haloferax volcanii adopts two distinct states under low ionic condition. One of these is similar to the β-grasp structure conserved in ubiquitin-like proteins from eukaryotes; the other is disordered, like prokaryotic ubiquitin-like protein, Pup. Furthermore, our study reveals that the conformation of SAMP2 is dependent on ionic strength. With the increase of ion concentration, SAMP2 undergoes a conformational conversion from disorder to order, indicating that the ordered conformation is the functional form of SAMP2 under the physiological condition of H. volcanii.

Characterization of an archaeal ubiquitin‐like protein SAMP1

Small archaeal modifier protein (SAMP1) structurally resembles the eukaryotic ubiquitin (Ub) and bacterial MoaD, sharing with them the β‐grasp fold and C‐terminal GlyGly motif. Our study was aimed to understand the function of SAMP1 using the haloarchaeon Haloferax volcanii as a model system. We demonstrated in vitro that an archaeal homolog of the ubiquitin‐activating enzyme E1 (named ubiquitin‐like activating enzyme of archaea or UbaA) activates SAMP1 in an ATP‐ or GTP‐dependent manner. LC‐MS/MS analysis shows that SAMP1, like Ub, is conjugated to the Lys of its protein substrates by a mechanism requiring UbaA. Cleavage of SAMP1‐conjugates by a JAB1/MPN/MOV34 protease (HvJAMM1) provides evidence that sampylation is reversible. SAMP1 is also found essential for MoCo‐dependent dimethyl sulfoxide reductase activity, suggesting that SAMP1 functions similarly with MoaD in MoCo biosynthesis. Overall, the shared functional similarities of SAMP1 with Ub and MoaD provide a physiological framework in understanding the evolutionary relationship between two divergent biochemical pathways: protein conjugation and sulfur transfer. This work is funded by NIH (ROI GM057498) and DOE (DE‐FG02–05ER1560).

Archaeal JAB1/MPN/MOV34 metalloenzyme (HvJAMM1) cleaves ubiquitin‐like small archaeal modifier proteins (SAMPs) from protein‐conjugates

Proteins with JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains are widespread among all domains of life, yet poorly understood. Here we report the purification and characterization of an archaeal JAMM/MPN+ domain protein (HvJAMM1) from Haloferax volcanii that cleaves ubiquitin-like small archaeal modifier proteins (SAMP1/2) from protein conjugates. HvJAMM1 cleaved SAMP1/2 conjugates generated in H. volcanii as well as isopeptide- and linear-linked SAMP1-MoaE in purified form. Cleavage of linear linked SAMP1-MoaE was dependent on the presence of the SAMP domain and the C-terminal VSGG motif of this domain. While HvJAMM1 was inhibited by size exclusion chromatography and metal chelators, its activity could be restored by addition of excess ZnCl2 . HvJAMM1 residues (Glu31, His88, His90, Ser98 and Asp101) that were conserved with the JAMM/MPN+ active-site motif were required for enzyme activity. Together, these results provide the first example of a JAMM/MPN+ zinc metalloprotease that independently catalyses the cleavage of ubiquitin-like (isopeptide and linear) bonds from target proteins. In archaea, HvJAMM1 likely regulates sampylation and the pools of 'free' SAMP available for protein modification. HvJAMM1-type proteins are thought to release the SAMPs from proteins modified post-translationally as well as those synthesized as domain fusions.

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.

Proteasomes and protein conjugation across domains of life.

Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes.

Uncovering ubiquitin and ubiquitin-like signaling networks.

Proteomes are significantly more complex than genomes and transcriptomes due to protein processing and extensive post-translational modification (PTM) of proteins. Hundreds of different modifications exist. Release 66 of the RESID database(1) (http://www.ebi.ac.uk/RESID/) contains 559 different modifications, including small chemical modifications such as phosphorylation, acetylation, and methylation and modification by small proteins, including ubiquitin and ubiquitin-like (UBL) proteins that are covalently coupled to proteins to regulate their activity. A wide variety of cellular processes are regulated by these reversible modifications, including transcription, replication, cell-cycle progression, and responses to DNA damage. Protein modifications have been studied for many years at the level of single target proteins, but currently available technologies enable proteome-wide studies of these modifications by mass spectrometry (MS).2,3 Powerful proteomics tools are available to study phosphorylation and acetylation at a systems-wide level in a site-specific manner. It is more challenging to study ubiquitin targets and targets for ubiquitin-like proteins at a proteome-wide level in a site-specific manner due to the relatively large size of these modifications, but hundreds of potential target proteins have been uncovered over the past eight years, mainly in a non-site-specific manner. This review is focused on uncovering signaling networks for ubiquitin and ubiquitin-like proteins by mass spectrometry and highlights the site-specific studies published in 2010 and 2011. Site-specific methodologies will likely have a major impact on the ubiquitin field in the near future. The methodology, results, challenges, pitfalls, crosstalk with other PTMs, and future directions are discussed in this review. 1.1. Ubiquitin and Ubiquitin-like Proteins Ubiquitin was first discovered in the mid-1970s, and the 2004 Nobel Prize in Chemistry was awarded for this finding. Ubiquitin is a 76 amino acid protein that is highly conserved from yeast to plants and mammals. Many ubiquitin-like proteins have been uncovered, including Nedd8, small ubiquitin-like modifier 1 (SUMO-1), SUMO-2, SUMO-3, FUBI, HUB1, ISG15, FAT10, URM1, UFM1, Atg12, and Atg8. Ubiquitin-like proteins are also found in prokaryotes and archaea; PUPs are prokaryotic ubiquitin-like proteins, and SAMPs are ubiquitin-like small archaeal modifier proteins. Despite limited sequence homology of some family members with ubiquitin, all ubiquitin family members display structural homology via the characteristic β-grasp ubiquitin fold.4−9 These small proteins are covalently coupled to target proteins via isopeptide bonds between C-terminal diglycine motifs and e-amino groups in lysines of target proteins using an enzymatic cascade that consists of an E1 enzyme,(10) an activator of ubiquitin and UBLs, an E2 enzyme,11,12 and a ligase, known as an E3 enzyme(13) (Figure ​(Figure1).1). Humans express 8 E1 enzymes(10) (including 1 dedicated to ubiquitin, 1 shared between ubiquitin and the UBL FAT10, and 6 dedicated to other UBLs) and 35 active E2 enzymes (including 28 dedicated to ubiquitin, 3 shared between ubiquitin and the UBL ISG15, 3 dedicated to other UBLs, and 1 putative E2).(12) Ubiquitin E3 enzymes are subdivided into HECT-type E3 enzymes (homology to E6AP carboxyl terminus)(14) and RING-type E3 enzymes (really interesting new gene).(15) HECT-type E3 enzymes form thioesters with ubiquitin, whereas RING-type E3 enzymes lack catalytic cysteines. Over 600 human genes encode components of RING-based E3 ligases.(15) Figure 1 Ubiquitylation cascade. Ubiquitin precursors are processed by proteases to generate mature ubiquitin containing a C-terminal diglycine motif for conjugation to target proteins. Three different classes of enzymes are involved: E1, E2, and E3 enzymes. Ubiquitin ...

Solution structure of the small archaeal modifier protein 1 (SAMP1) from Methanosarcina acetivorans.

Solution structure of the small archaeal modifier protein 1 (SAMP1) from Methanosarcina acetivorans.

Crystal structure of ubiquitin-like small archaeal modifier protein 1 (SAMP1) from Haloferax volcanii

The ubiquitin-like (Ubl) system has been shown to be ubiquitous in all three kingdoms of life following the very recent characterization of ubiquitin-like small archaeal modifier proteins (SAMP1 and 2) from Haloferax volcanii. The ubiquitin (Ub) and Ubl molecules in eukaryotes have been studied extensively and their cellular functions are well established. Biochemical and structural data pertaining to prokaryotic Ubl protein (Pup) continue to be reported. In contrast to eukaryotes and prokaryotes, no structural information on the archaeal Ubl molecule is available. Here we determined the crystal structure of SAMP1 at 1.55 Å resolution and generated a model of SAMP2. These were then compared with other Ubl molecules from eukaryotes as well as prokaryotes. The structure of SAMP1 shows a β-grasp fold of Ub, suggesting that the archaeal Ubl molecule is more closely related to eukaryotic Ub and Ubls than to its prokaryotic counterpart. The current structure identifies the location of critical elements such a single lysine residue (Lys4), C-terminal di-glycine motif, hydrophobic patches near leucine 60, and uniquely inserted α-helical segments (α1 and α3) in SAMP1. Based on the structure of SAMP1, several Ub-like features of SAMPs such as poly-SAMPylation and non-covalent interactions have been proposed, which should provide the basis for further investigations concerning the molecular function of archaeal Ubls and the large super-family of β-grasp fold proteins in the archaeal kingdom.