Structural basis of purine nucleotide inhibition of human uncoupling protein 1.

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.

Structural and functional characterization of Cas2 of CRISPR-Cas subtype I-C lacking the CRISPR component.

The genome of pathogenic Leptospira interrogans serovars (Copenhageni and Lai) are predicted to have CRISPR-Cas of subtypes I-B and I-C. Cas2, one of the core Cas proteins, has a crucial role in adaptive defense against foreign nucleic acids. However, subtype I-C lacks the CRISPR element at its loci essential for RNA-mediated adaptive immunity against foreign nucleic acids. The reason for sustaining the expense of cas genes are unknown in the absence of a CRISPR array. Thus, Cas2C was chosen as a representative Cas protein from two well-studied serovars of Leptospira to address whether it is functional. In this study, the recombinant Cas2C of Leptospira serovars Copenhageni (rLinCas2C, 12 kDa) and Lai (rLinCas2C_Lai, 8.6 kDa) were overexpressed and purified. Due to natural frameshift mutation in the cas2c gene of serovar Lai, rLinCas2C_Lai was overexpressed and purified as a partially translated protein. Nevertheless, the recombinant Cas2C from each serovar exhibited metal-dependent DNase and metal-independent RNase activities. The crystal structure of rLinCas2C obtained at the resolution of 2.60 Å revealed the protein is in apostate conformation and contains N- (1-71 amino acids) and C-terminal (72-90 amino acids) regions, with the former possessing a ferredoxin fold. Substitution of the conserved residues (Tyr7, Asp8, Arg33, and Phe39) with alanine and deletion of Loop L2 resulted in compromised DNase activity. On the other hand, a moderate reduction in RNase activity was evident only in selective rLinCas2C mutants. Overall, in the absence of an array, the observed catalytic activity of Cas2C may be required for biological processes distinct from the CRISPR-Cas-associated function.

Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes.

Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.

Water-mediated structural rearrangement establishes active conformation of caspases for apoptosis and inflammation.

Caspases are cysteine-dependent aspartate-specific proteases that play a crucial role in apoptosis (or programmed cell death) and inflammation. Based on their function, caspases are majorly categorized into apoptotic (initiator/apical and effector/executioner) and inflammatory caspases. Caspases undergo transition from an inactive zymogen to an active caspase to accomplish their function. This transition demands structural rearrangements which are most prominent at the active site loops and are imperative for the catalytic activity of caspases. In effector caspase-3, the structural rearrangement in the active site loop is shown to be facilitated by a set of invariant water (IW) molecules. However, the atomic details involving their role in stabilizing the active conformation have not been reported yet. Moreover, it is not known whether water molecules are essential for the active conformation in all caspases. Thus, in this study, we located IW molecules in initiator, effector, and inflammatory caspases to understand their precise role in rendering the structural arrangement of active caspases. Furthermore, IW molecules involved in anchoring the fragments of the protomer and rendering regulated flaccidity to caspases were identified. Location and identification of IW molecules interacting with amino acid residues involved in establishing the active conformation in the caspases might facilitate the design of potent inhibitors during up-regulated caspase activity in neurodegenerative and immune disorders. Communicated by Ramaswamy H. Sarma.

Structural and functional role of invariant water molecules in matrix metalloproteinases: a data-mining approach.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases known to degrade extracellular matrix (ECM). Being involved in many biological and physiological processes of tissue remodeling, MMPs play a crucial role in many pathological conditions such as arthritis, cancer, cardiovascular diseases, etc. Typically, MMPs possess a propeptide, a zinc-containing catalytic domain, a hinge region and a hemopexin domain. Based on their structural domain organization and substrates, MMPs are classified into six different classes, viz. collagenases, stromelysins, gelatinases, matrilysins, membrane-type and other MMPs. As per previous studies, a set of invariant water (IW) molecules of MMP-1 (a collagenase) play a significant role in stabilizing their catalytic domain. However, a functional role of IW molecule in other classes of MMPs has not been reported yet. Thus, in this study, IW molecules of MMPs from different classes were located and their plausible role(s) have been assigned. The results suggest that IW molecules anchor the structurally and functionally essential metal ions present in the vicinity of the active site of MMPs. Further, they (in)directly interlink different structural features and bridge the active site metal ions of MMPs. This study provides the key IW molecules that are structurally and functionally relevant to MMPs and hence, in turn, might facilitate the development of potent generalized inhibitor(s) against different classes of MMPs. Communicated by Ramaswamy H. Sarma.

The core Cas1 protein of CRISPR-Cas I-B in Leptospira shows metal-tunable nuclease activity.

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 is the causative agent of leptospirosis in animals and humans. This organism carries a functional cas1 gene classified under CRISPR-Cas I-B. In this study, using various nuclease assays and bioinformatics analysis, we report that the recombinant Cas1 (LinCas1) possesses metal-ion dependent DNase activity, which is inhibited upon substitution or chelation of metal-ion and/or interaction with recombinant Cas2 (LinCas2) of L. interrogans. Model of LinCas1 structure shows a shorter N-terminal domain unlike other Cas1 orthologs reported to date. The C-terminal domain of LinCas1 contains conserved divalent-metal binding residues (Glu108, His176, and Glu191) and the mutation of these residues leads to abolition in DNase activity. Immunoassay using anti-LinCas2 demonstrates that LinCas1 interacts with LinCas2 and attains a saturation point. Moreover, the nuclease activity of the LinCas1-Cas2 mixture on ds-DNA displayed a reduction in activity compared to the pure core LinCas proteins under in vitro condition. The DNase activity for LinCas1 is consistent with a role for this protein in the recognition/cleavage of foreign DNA and integration of foreign DNA as spacer into the CRISPR array.

Exploiting the rationale behind substrate recognition by promiscuous thermophilic NDP-sugar pyrophosphorylase for expanding glycorandomization: an in silico study.

The fundamental substrates for protein glycosylation are provided by a group of enzymes known as NDP-sugar pyrophosphorylases (NSPases) which utilize nucleotide triphosphate (NTP) and sugar 1-phosphate to catalyze the formation of nucleotide diphospho-sugar (NDP-sugar). The promiscuous nature of NSPases is often exploited during chemoenzymatic glycorandomization in the pursuit of novel therapeutics. However, till date, the number of inherently promiscuous NSPases reported and the rationale behind their promiscuity is meager. In this study, we have identified a set of NSPases from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 to identify probable candidates for glycorandomization. We identified a set of NSPases that include both substrate-specific and substrate-promiscuous NSPases with a visible predominance of the latter group. The rationale behind the promiscuity (or specificity) vividly lies in the repertoire of amino acid residues that assemble the active site for recognition of the substrate moiety. Furthermore, the absence of a function-specific auxiliary domain promotes substrate promiscuity in NSPases. This study, thus, provides a novel set of thermophilic NSPases that can be employed for chemoenzymatic glycorandomization. More importantly, identification of the residues that render substrate promiscuity (or specificity) would assist in sequence-based rational engineering of NSPases for enhanced glycorandomization. Communicated by Ramaswamy H. Sarma.

Structural and thermodynamic correlation illuminates the selective transport mechanism of disaccharide α-glycosides through ABC transporter.

Carbohydrate (or sugar) molecules are extremely diverse regarding their length, linkage and epimeric state. Selective acquisition of these molecules inside the cell is achieved by the substrate (or solute)-binding protein of ATP-binding cassette (ABC) transport system. However, the molecular mechanism underlying the selective transport of diverse carbohydrates remains unclear mainly owing to their structural complexity and stereochemistry. This study reports crystal structures of an α-glycoside-binding protein (αGlyBP, ORF ID: TTHA0356 from Thermus thermophilus HB8) in complex with disaccharide α-glycosides namely trehalose (α-1,1), sucrose (α-1,2), maltose (α-1,4), palatinose (α-1,6) and glucose within a resolution range of 1.6-2.0 Å. Despite transporting multiple types of sugars, αGlyBP maintains its stereoselectivity for both glycosidic linkage as well as an epimeric hydroxyl group. Out of the two subsites identified in the active-site pocket, subsite B which accommodates the glucose and glycosyl unit of disaccharide α-glycosides is highly conserved. In addition, structural data confirms the paradoxical behavior of glucose, where it replaces the high-affinity ligand(s) (disaccharide α-glycosides) from the active site of the protein. Comparative assessment of open and closed conformations of αGlyBP along with mutagenic and thermodynamic studies identifies the hinge region as the first interaction site for the ligands. On the other hand, encapsulation of ligand inside the active site is achieved through the N-terminal domain (NTD) movement, whereas the C-terminal domain (CTD) of αGlyBP is identified to be rigid and postulated to be responsible for maintaining the interaction with the transmembrane domain (TMD) during substrate translocation. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession number(s) 6J9W, 6J9Y, 6JAD, 6JAG, 6JAH, 6JAI, 6JAL, 6JAM, 6JAN, 6JAO, 6JAP, 6JAQ, 6JAR, 6JAZ, 6JB0, 6JB4, 6JBA, 6JBB and 6JBE.

Design of novel PhMTNA inhibitors, targeting neurological disorder through homology modeling, molecular docking, and dynamics approaches.

Vanishing white matter (VWM) is a hereditary human disease, mostly prevalent in childhood caused by the defects in the eukaryotic initiation factor beta subunits. It is the first disease involved in the translation initiation factor, eIF2B. There is no specific treatment for VWM which mainly affect the brain and ovaries. The gray matter remains normal in all characteristics while the white matter changes texture, coming to the pathophysiology, many initiation factors are involved in the initiation of translation of mRNAs into polypeptides. In this study, the three-dimensional structure of PhMTNA protein was modeled and the stability ascertained through Molecular dynamic simulation (MDS) for 100 ns. The active site residues are conserved with the reported BsMTNA structure which is also confirmed through sitemap prediction. Through virtual screening and induced fit docking, top five leads against PhMTNA protein was identified based on their binding mode and affinity. ADME properties and DFT (Density Functional Theory) studies of these compounds were studied. In addition to that, computational mutagenesis studies were performed to identify the hotspot residues involved in the protein-ligand interactions. Overall analysis showed that the compound NCI_941 has a highest binding energy of -46.256 kcal mol-1 in the Arg57Ala mutant. Thus, the results suggest that NCI_941 would act as a potent inhibitor against PhMTNA protein.

Role of Structural Features in Oligomerization, Active-Site Integrity and Ligand Binding of Ribose-1,5-Bisphosphate Isomerase.

Pentose bisphosphate pathway, exclusively found in archaea, is similar to the pentose phosphate pathway present in bacteria and eukarya. In pentose bisphosphate pathway, the conversion of ribose moieties of nucleosides into 3-phosphoglycerate (3-PGA) involves multiple steps; one of them being the conversion of ribose-1,5-bisphosphate (R15P) to ribulose-1,5-bisphosphate (RuBP) catalyzed by an enzyme ribose-1,5-bisphosphate isomerase (R15Pi). The availability of the three-dimensional structure of R15Pi had facilitated the understanding of various structural and functional aspects of the enzyme. Nevertheless, the structure of R15Pi also left several significant questions unanswered that would aid in understanding the structure-function relationship of the enzyme. Thus, we have taken up a computational approach to further understand the role of various structural features of the enzyme R15Pi. Results obtained from molecular dynamics (MD) simulations aided in understanding the obligation of the enzyme R15Pi to oligomerize and also in deciphering the role of catalytic residue(s) in structural stability. Identification of invariant water molecules of the enzyme R15Pi helped in discerning their significance at the active-site pocket and structurally important regions. Further, molecular docking studies allowed the identification of the amino acid residues essential for holding the substrate (R15P) or product (RuBP) in the vicinity of the active site of the enzyme R15Pi. Interestingly, results of the molecular docking studies assisted in the identification of an "alternate binding site" for the substrate, R15P. Finally, based on these results, we propose a mechanism of "substrate sliding" for the enzyme R15Pi.

Designating ligand specificities to metal uptake ABC transporters in Thermus thermophilus HB8.

Micronutrients such as metal ions are indispensable for the growth and survival of microorganisms in assorted environmental niches. However, change in cellular concentration of metal ions is pernicious for an organism; thus metal ion homeostasis is crucial for their survival and growth. An eminent mechanism for maintaining metal ion homeostasis in microorganisms is ATP-binding cassette (ABC) transporters, which transport metal ions in their ionic/complex forms across the cell membrane. For the uptake, metals are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains (TMDs) for their transport. In this work, a high-throughput data mining analysis has been performed to identify open reading frames (ORFs) encoding metal-specific ABC transporters in a thermophilic bacterium Thermus thermophilus HB8. In total, 22 ORFs resulting in eight ABC transport systems were identified, which are potentially involved in the uptake of metal ions. This study suggests that three out of eight metal-specific ABC import systems are specific to iron ions. Among the remaining five, two are particular to divalent metal ions such as Mg2+ and Zn2+/Mn2+, another two are for tetrahedral oxyanions such as MoO42- and WO42- and the remaining one imports cyanocobalamin (vitamin B12). Besides these, the results of this study demonstrate the existence of a mechanism where TMD and NBD components are shared among different ABC transport systems hinting that multiple substrates can be imported via a single transporter. This study thus provides the first ever preliminary glimpse into the entire repertoire of metal uptake ABC transporters in a thermophilic organism.

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase.

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.

Archaeal and eukaryal translation initiation factor 1 differ in their RNA interacting loops.

The archaeal translation initiation factor 1 (aIF1) is reported to be functionally homologous to the eukaryotic translation initiation factor 1 (eIF1). However, lack of a structural comparison between aIF1 and eIF1 has limited our understanding of the structural (dis)similarities. Herein, we have determined the three-dimensional crystal structure of an open reading frame PH1771.1 encoding aIF1 in Pyrococcus horikoshii OT3. Results reveal that although aIF1 has low sequence similarity with eIF1, high structural homology exists between the two proteins. Nonetheless, notable critical differences between aIF1 and eIF1 could still be perceived at the ß1 -ß2 basic loop, the acidic loop and the solvent-exposed surface. These differences might lead to a slightly divergent mode of action of aIF1 during archaeal translation initiation.

A presumed homologue of the regulatory subunits of eIF2B functions as ribose-1,5-bisphosphate isomerase in Pyrococcus horikoshii OT3.

The homologues of the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B) are assumed to be present in archaea. Likewise, an ORF, PH0208 in Pyrococcus horikoshii OT3 have been proposed to encode one of the homologues of regulatory subunits of eIF2B. However, PH0208 protein also shares sequence similarity with a functionally non-related enzyme, ribose-1,5-bisphosphate isomerase (R15Pi), involved in conversion of ribose-1,5-bisphosphate (R15P) to ribulose-1,5-bisphosphate (RuBP) in an AMP-dependent manner. Herein, we have determined the crystal structure of PH0208 protein in order to decipher its true function. Although structurally similar to the regulatory subunits of eIF2B, the ability to bind R15P and RuBP suggests that PH0208 would function as R15Pi. Additionally, this study for the first time reports the binding sites of AMP and GMP in R15Pi. The AMP binding site in PH0208 protein clarified the role of AMP in providing structural stability to R15Pi. The binding of GMP to the 'AMP binding site' in addition to its own binding site indicates that GMP might also execute a similar function, though with less specificity. Furthermore, we have utilized the resemblance between PH0208 and the regulatory subunits of eIF2B to propose a model for the regulatory mechanism of eIF2B in eukaryotes.

Computational characterization of TTHA0379: A potential glycerophosphocholine binding protein of Ugp ATP-binding cassette transporter.

For the de novo biosynthesis of phospholipids, byproducts such as sn-glycerol-3-phosphate (G3P) and glycerophosphocholine (GPC) of glycerophospholipid metabolic pathway are imported inside the cell by an ATP-binding cassette (ABC) transporter known as UgpABCE. Of which, UgpA and UgpE constitutes the transmembrane domains (TMDs), UgpC forms the dimer of ATP-hydrolyzing component and UgpB is the periplasmic substrate binding protein. Structurally, UgpABCE transporter displays similarity to the maltose ABC transporter of Escherichia coli; thus, has been grouped into the CUT1 (Carbohydrate Uptake Transporter-1) family of bacterial ABC transporters. Being a member of CUT1 family, several Ugp (Uptake glycerol phosphate) protein sequences in biological database(s) exhibit sequence and structure similarity to sugar ABC transporters and have been annotated as sugar binding proteins; one of such proteins is TTHA0379 from Thermus thermophilus HB8. Here, in this study, we used computational method(s) to distinguish UgpB and sugar binding proteins based on their primary and tertiary structure features. A comprehensive analysis of these proteins indicates that they are evolutionarily related to each other having common conserved features at their primary and tertiary structure levels. However, they display differences at their active sites owing to the dissimilarity in their ligand preferences. In addition, phylogenetic analysis of TTHA0379 along with UgpB and sugar binding proteins reveals that both the groups of proteins forms two distinct clades and TTHA0379 groups with UgpB proteins. Furthermore, analysis of the ligand binding pocket shows that all the essential features of glycerophosphocholine binding protein i.e. UgpB, are conserved in TTHA0379 as well. Combining these features, here, we designate TTHA0379 to be a GPC binding protein.

In silico analysis of 5'-UTRs highlights the prevalence of Shine-Dalgarno and leaderless-dependent mechanisms of translation initiation in bacteria and archaea, respectively.

In prokaryotes, a heterogeneous set of protein translation initiation mechanisms such as Shine-Dalgarno (SD) sequence-dependent, SD sequence-independent or ribosomal protein S1 mediated and leaderless transcript-dependent exists. To estimate the distribution of coding sequences employing a particular translation initiation mechanism, a total of 107 prokaryotic genomes were analysed using in silico approaches. Analysis of 5'-untranslated regions (UTRs) of genes reveals the existence of three types of mRNAs described as transcripts with and without SD motif and leaderless transcripts. Our results indicate that although all the three types of translation initiation mechanisms are widespread among prokaryotes, the number of SD-dependent genes in bacteria is higher than that of archaea. In contrast, archaea contain a significantly higher number of leaderless genes than SD-led genes. The correlation analysis between genome size and SD-led & leaderless genes suggests that the SD-led genes are decreasing (increasing) with genome size in bacteria (archaea). However, the leaderless genes are increasing (decreasing) in bacteria (archaea) with genome size. Moreover, an analysis of the start-codon biasness confirms that among ATG, GTG and TTG codons, ATG is indeed the most preferred codon at the translation initiation site in most of the coding sequences. In leaderless genes, however, the codons GTG and TTG are also observed at the translation initiation site in some species contradicting earlier studies which suggested the usage of only ATG codon. Henceforth, the conventional mechanism of translation initiation cannot be generalized as an exclusive way of initiating the process of protein biosynthesis in prokaryotes.

Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system.

Heterogeneous behavior of metalloproteins toward metal ion binding and selectivity: insights from molecular dynamics studies.

About one-third of the existing proteins require metal ions as cofactors for their catalytic activities and structural complexities. While many of them bind only to a specific metal, others bind to multiple (different) metal ions. However, the exact mechanism of their metal preference has not been deduced to clarity. In this study, we used molecular dynamics (MD) simulations to investigate whether a cognate metal (bound to the structure) can be replaced with other similar metal ions. We have chosen seven different proteins (phospholipase A2, sucrose phosphatase, pyrazinamidase, cysteine dioxygenase (CDO), plastocyanin, monoclonal anti-CD4 antibody Q425, and synaptotagmin 1 C2B domain) bound to seven different divalent metal ions (Ca(2+), Mg(2+), Zn(2+), Fe(2+), Cu(2+), Ba(2+), and Sr(2+), respectively). In total, 49 MD simulations each of 50 ns were performed and each trajectory was analyzed independently. Results demonstrate that in some cases, cognate metal ions can be exchanged with similar metal ions. On the contrary, some proteins show binding affinity specifically to their cognate metal ions. Surprisingly, two proteins CDO and plastocyanin which are known to bind Fe(2+) and Cu(2+), respectively, do not exhibit binding affinity to any metal ion. Furthermore, the study reveals that in some cases, the active site topology remains rigid even without cognate metals, whereas, some require them for their active site stability. Thus, it will be interesting to experimentally verify the accuracy of these observations obtained computationally. Moreover, the study can help in designing novel active sites for proteins to sequester metal ions particularly of toxic nature.

In silico analysis suggests that PH0702 and PH0208 encode for methylthioribose-1-phosphate isomerase and ribose-1,5-bisphosphate isomerase, respectively, rather than aIF2Bß and aIF2Bδ.

The overall process of protein biosynthesis across all domains of life is similar; however, detailed insights reveal a range of differences in the proteins involved. For decades, the process of protein translation in archaea has been considered to be closer to eukaryotes than to bacteria. In archaea, however, several homologues of eukaryotic proteins involved in translation initiation have not yet been identified; one of them being the initiation factor eIF2B consisting of five subunits (α, ß, γ, δ and ε). Three open reading frames (PH0440, PH0702 and PH0208) in Pyrococcus horikoshii have been proposed to encode for the α-, ß- and δ-subunits of aIF2B, respectively. The crystal structure of PH0440 shows similarity toward the α-subunit of eIF2B. However, the capability of PH0702 and PH0208 to function as the ß- and δ-subunits of eIF2B, respectively, remains uncertain. In this study, we have taken up the task of annotating PH0702 and PH0208 using bioinformatics methods. The phylogenetic analysis of protein sequences belonging to IF2B-like family along with PH0702 and PH0208 revealed that PH0702 belonged to methylthioribose-1-phosphate isomerase (MTNA) group of proteins, whereas, PH0208 was found to be clustered in the group of ribose-1,5-bisphosphate isomerase (R15PI) proteins. A careful analysis of protein sequences and structures available for eIF2B, MTNA and R15PI confirms that PH0702 and PH0208 contain residues essential for the enzymatic activity of MTNA and R15PI, respectively. Additionally, the protein PH0208 comprises of the residues required for the dimer formation which is essential for the biological activity of R15PI. This prompted us to examine all eIF2B-like proteins from archaea and to annotate their function. The results reveal that majority of these proteins are homologues of the α-subunit of eIF2B, even though they lack the residues essential for their functional activity. A better understanding of the mechanism of GTP exchange during translation initiation in archaea is henceforth required.