hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50.

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

Conserved features of the MlaD domain aid the trafficking of hydrophobic molecules.

In Gram-negative bacteria, the maintenance of lipid asymmetry (Mla) system is involved in the transport of phospholipids between the inner (IM) and outer membrane. The Mla system utilizes a unique IM-associated periplasmic solute-binding protein, MlaD, which possesses a conserved domain, MlaD domain. While proteins carrying the MlaD domain are known to be primarily involved in the trafficking of hydrophobic molecules, not much is known about this domain itself. Thus, in this study, the characterization of the MlaD domain employing bioinformatics analysis is reported. The profiling of the MlaD domain of different architectures reveals the abundance of glycine and hydrophobic residues and the lack of cysteine residues. The domain possesses a conserved N-terminal region and a well-preserved glycine residue that constitutes a consensus motif across different architectures. Phylogenetic analysis shows that the MlaD domain archetypes are evolutionarily closer and marked by the conservation of a functionally crucial pore loop located at the C-terminal region. The study also establishes the critical role of the domain-associated permeases and the driving forces governing the transport of hydrophobic molecules. This sheds sufficient light on the structure-function-evolutionary relationship of MlaD domain. The hexameric interface analysis reveals that the MlaD domain itself is not a sole player in the oligomerization of the proteins. Further, an operonic and interactome map analysis reveals that the Mla and the Mce systems are dependent on the structural homologs of the nuclear transport factor 2 superfamily.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.

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.

An updated classification and mechanistic insights into ligand binding of the substrate-binding proteins.

Substrate-binding proteins (SBPs) mediate ligand translocation and have been classified into seven clusters (A-G). Although the substrate specificities of these clusters are known to some extent, their ligand-binding mechanism(s) remain(s) incompletely understood. In this study, the list of SBPs belonging to different clusters was updated (764 SBPs) compared to the previously reported study (504 SBPs). Furthermore, a new cluster referred to as cluster H was identified. Results reveal that SBPs follow different ligand-binding mechanisms. Intriguingly, the majority of the SBPs follow the 'one domain movement' rather than the well-known 'Venus Fly-trap' mechanism. Moreover, SBPs of a few clusters display subdomain conformational movement rather than the complete movement of the N- and C-terminal domains.

Structural and thermodynamic insights into the novel dinucleotide-binding protein of ABC transporter unveils its moonlighting function.

Substrate (or solute)-binding proteins (SBPs) selectively bind the target ligands and deliver them to the ATP-binding cassette (ABC) transport system for their translocation. Irrespective of the different types of ligands, SBPs are structurally conserved. A wealth of structural details of SBPs bound to different types of ligands and the physiological basis of their import are available; however, the uptake mechanism of nucleotides is still deficient. In this study, we elucidated the structural details of an SBP endogenously bound to a novel ligand, a derivative of uridylyl-3'-5'-phospho-guanosine (U3G); thus, we named it a U3G-binding protein (U3GBP). To the best of our knowledge, this is the first report of U3G (and a dinucleotide) binding to the SBP of ABC transport system, and thus, U3GBP is classified as a first member of subcluster D-I SBPs. Thermodynamic data also suggest that U3GBP can bind phospholipid precursor sn-glycerophosphocholine (GPC) at a site other than the active site. Moreover, a combination of mutagenic and structural information reveals that the protein U3GBP follows the well-known 'Venus Fly-trap' mechanism for dinucleotide binding. DATABASES: Structural data are available in RCSB Protein Data Bank under the accession number(s) 7C0F, 7C0K, 7C0L, 7C0O, 7C0R, 7C0S, 7C0T, 7C0U, 7C0V, 7C0W, 7C0X, 7C0Y, 7C0Z, 7C14, 7C15, 7C16, 7C19, and 7C1B.

Conformational Trapping of a ß-Glucosides-Binding Protein Unveils the Selective Two-Step Ligand-Binding Mechanism of ABC Importers.

Substrate-binding proteins (SBPs), selectively capture ligand(s) and ensure their translocation via its cognate ATP-binding cassette (ABC) import system. SBPs bind their cognate ligand(s) via an induced-fit mechanism known as the "Venus Fly-trap"; however, this mechanism lacks the atomic details of all conformational landscape as the confirmatory evidence(s) in its support. In this study, we delineate the atomic details of an SBP, ß-glucosides-binding protein (ßGlyBP) from Thermus thermophilus HB8. The protein ßGlyBP is multi-specific and binds to different types of ß-glucosides varying in their glycosidic linkages viz. ß-1,2; ß-1,3; ß-1,4 and ß-1,6 with a degree of polymerization of 2-5 glucosyl units. Structurally, the protein ßGlyBP possesses four subdomains (N1, N2, C1 and C2). The unliganded protein ßGlyBP remains in an open state, which closes upon binding to sophorose (SOP2), laminari-oligosaccharides (LAMn), cello-oligosaccharides (CELn), and gentiobiose (GEN2). This study reports, for the first time, four different structural states (open-unliganded, partial-open-unliganded, open-liganded and closed-liganded) of the protein ßGlyBP, revealing its conformational changes upon ligand binding and suggesting a two-step induced-fit mechanism. Further, results suggest that the conformational changes of N1 and C1 subdomains drive the ligand binding, unlike that of the whole N- and C-terminal domains (NTD and CTD) as known in the "Venus Fly-trap" mechanism. Additionally, profiling of stereo-selection mechanism for α- and ß-glucosides reveals that in the ligand-binding site four secondary structural elements (L1, H1, H2 and H3) drive the ligand selection. In summary, results demonstrate that the details of conformational changes and ligand selection are pre-encoded in the SBPs.

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.

Identification and characterization of ABC transporters for carbohydrate uptake in Thermus thermophilus HB8.

Organisms use a variety of carbohydrates and metabolic pathways in order to capitalize in their specific environments. Depending upon their habitat, organism employs different types of transporters to maintain the cellular nutritional balance via central metabolism. A major contributor in this process in bacteria is a carbohydrate ABC transporter. The focus of this study is to get an insight into the carbohydrate transport and metabolism of a hot-spring-dwelling bacterium Thermus thermophilus HB8. We applied high-throughput data-mining approaches for identification and characterization of carbohydrate ABC transporters in T. thermophilus HB8. This enabled the identification of 11 putative carbohydrate ABC transport systems. To identify the cognate ligands for these transporters, functional annotation was performed. However, scarcity of homologous-protein's function hinders the process of functional annotation. Thus, to overcome this limitation, we integrated the functional annotation of carbohydrate ABC transporters with their metabolic analysis. Our results demonstrate that out of 11 putative carbohydrate ABC transporters, six are involved in the sugar (four for monosaccharides and polysaccharides-degraded products and two for osmotic regulation), four in phospholipid precursor (namely UgpABCE) and the remaining one in purine uptake. Further, analysis suggests the existence of sharing mechanism of transmembrane domains (TMDs) and/or nucleotide-binding domains (NBDs) among the 11 carbohydrate ABC transporters.

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.

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.

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.