Academic literature on the topic 'Domain-architecture specific residues'

ABSTRACT CTnDOT integrase (IntDOT) is a member of the tyrosine family of site-specific DNA recombinases. IntDOT is unusual in that it catalyzes recombination between nonidentical sequences. Previous mutational analyses centered on mutants with substitutions of conserved residues in the catalytic (CAT) domain or residues predicted by homology modeling to be close to DNA in the core-binding (CB) domain. That work suggested that a conserved active-site residue (Arg I) of the CAT domain is missing and that some residues in the CB domain are involved in catalysis. Here we used a genetic approach and constructed an Escherichia coli indicator strain to screen for random mutations in IntDOT that disrupt integrative recombination in vivo. Twenty-five IntDOT mutants were isolated and characterized for DNA binding, DNA cleavage, and DNA ligation activities. We found that mutants with substitutions in the amino-terminal (N) domain were catalytically active but defective in forming nucleoprotein complexes, suggesting that they have altered protein-protein interactions or altered interactions with DNA. Replacement of Ala-352 of the CAT domain disrupted DNA cleavage but not DNA ligation, suggesting that Ala-352 may be important for positioning the catalytic tyrosine (Tyr-381) during cleavage. Interestingly, our biochemical data and homology modeling of the CAT domain suggest that Arg-285 is the missing Arg I residue of IntDOT. The predicted position of Arg-285 shows it entering the active site from a position on the polypeptide backbone that is not utilized in other tyrosine recombinases. IntDOT may therefore employ a novel active-site architecture to catalyze recombination.

Abstract Abstract 1148 Coagulation factor XI (FXI) is a plasma zymogen that is activated to FXIa, the catalytic domain of which contains exosites that interact with its normal macromolecular substrate (FIX), and its major regulatory inhibitor (protease nexin-2 kunitz protease inhibitor, PN2KPI). To localize the catalytic domain residues involved in active site architecture and in various ligand-binding exosites, we aligned the sequence of the FXI catalytic domain with that of the prekallikrein (PK) catalytic domain which is highly homologous (64% identity) in sequence, but functionally very different from FXI. Six distinct regions (R1-R6) of dissimilarity between the two proteins were identified as possible candidates for FXIa-specific ligand binding exosites. FXI/PK chimeric proteins (FXI-R1, FXI-R2, FXI-R3, FXI-R4, FXI-R5, and FXI-R6) containing substitutions with PK residues within the six regions were prepared and characterized. FXIa-R1, R2, R3 displayed enhanced proteolysis after activation suggesting that the residues within R1, R2 and R3 regions may be important to maintain proper folding of the enzyme. Comparisons of amidolytic assays vs. activated partial thromboplastin time assays showed similar activities for all chimeras except FXI-R6, which displayed 60% of the normal amidolytic activity but only 28% of clotting activity suggesting the possibility that the R6 region (autolysis loop) of FXIa may comprise an exosite involved in the interaction with its macromolecular substrate FIX. This hypothesis was further confirmed experiments showing that the proteolytic activation of FIX by FXIa-R6 was significantly impaired compared with that achieved by FXIawt. Although FXIa-R5 and FXIa-R6 were defective (50-60%) in amidolytic assays, these chimeras were very similar to FXIawt in heparin and high molecular weight kininogen binding assays, suggesting that residues within the R5 and R6 regions are involved in active-site architecture. These chimeras were further investigated to determine whether any of them had acquired kallikrein activity. After activation all except FXIa-R4 showed insignificant activity using a kallikrein-specific substrate. FXIa-R4 displayed 87% of the activity of kallikrein using the kallikrein-specific substrate but only 3% of the activity of FXIawt using the FXIa chromogenic substrate. Moreover the cleavage pattern and cleavage rate of high molecular weight kininogen by FXIa-R4 were similar to those achieved by kallikrein but not by FXIawt. Therefore substitutions in the R4 region of FXI with the corresponding residues of PK resulted in loss of activity for the FXIa substrates and gain of activity for the kallikrein substrates suggesting that the R4 region (99-loop) of FXIa plays a role in determining the substrate specificity. From the co-crystal structure of the FXIa catalytic domain with PN2KPI, the residues R3704, Y5901, E98, Y143, I151, and K192 (chymotrypsin numbering) in the FXIa catalytic domain have been identified to be possibly involved in the interactions with its inhibitors. A single mutation comprising Y5901A in the R2 region of FXIa does not affect folding however this mutant displayed resistance to inhibition by PN2KPI indicating that Y5901 is involved in the interaction of FXIa with PN2KPI. In conclusion, these studies of FXI/PK chimeric and mutant proteins implicate residues within the R4 region (99-loop) of FXIa in the determination of amidolytic substrate specificity; residues within the R6 region (autolysis loop) of FXIa in the interaction with the macromolecular substrate, FIX; and the residue Y5901 in the R2 region of FXIa in the interaction of FXIa with PN2KPI. Disclosures: No relevant conflicts of interest to declare.

Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond (CS-SC) preconditions. The simulations confirmed the major role of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central 54CS-S198C or 61CS-S192C contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to −45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable.

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro. Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

ABSTRACT Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. While the biological roles of the Ada, AlkA, and AlkB proteins have been defined, despite many efforts, the molecular functions of AidB remain largely unknown. In this study, we focused on the biological role of the AidB protein, and we demonstrated that AidB shows preferential binding to a DNA region that includes the upstream element of its own promoter, PaidB. The physiological significance of this specific interaction was investigated by in vivo gene expression assays, demonstrating that AidB can repress its own synthesis during normal cell growth. We also showed that the domain architecture of AidB is related to the different functions of the protein: the N-terminal region, comprising the first 439 amino acids (AidB “I-III”), possesses FAD-dependent dehydrogenase activity, while its C-terminal domain, corresponding to residues 440 to 541 (AidB “IV”), displays DNA binding activity and can negatively regulate the expression of its own gene in vivo. Our results define a novel role in gene regulation for the AidB protein and underline its multifunctional nature.

Kinetoplastea are free living and parasitic protists with unique features among Eukaryota. Pathogenic Kinetoplastea parasites (i.e., Trypanosoma and Leishmania spp.) undergo several developmental transitions essential for survival in their hosts. These transitions require membrane and cytoskeleton reorganizations that involve phosphoinositides (PIs). Phospholipids like PIs are key regulators of vital functions in all eukaryotes including signal transduction, protein transport and sorting, membrane trafficking, and cytoskeleton and membrane remodeling. A large repertoire of PI-metabolizing enzymes and PI-binding proteins/effectors carrying distinct PI-binding modules like the PX (phox homology) module could play significant roles in the life and virulence of pathogenic Kinetoplastea. The aim of this study was to retrieve the entire spectrum of Kinetoplastea protein sequences containing the PX module (PX-proteins), predict their structures, and identify in them evolutionary conserved and unique traits. Using a large array of bioinformatics tools, protein IDs from two searches (based on PFam’s pHMM for PX domain (PF00787)) were combined, aligned, and utilized for the construction of a new Kinetoplastea_PX pHMM. This three-step search retrieved 170 PX-protein sequences. Structural domain configuration analysis identified PX, Pkinase, Lipocalin_5, and Vps5/BAR3-WASP domains and clustered them into five distinct subfamilies. Phylogenetic tree and domain architecture analysis showed that some domain architectures exist in proteomes of all Kinetoplastea spp., while others are genus-specific. Finally, amino acid conservation logos of the Kinetoplastea spp. and Homo sapiens PX domains revealed high evolutionary conservation in residues forming the critical structural motifs for PtdIns3P recognition. This study highlights the PX-Pkinase domain architecture as unique within Trypanosoma spp. and forms the basis for a targeted functional analysis of Kinetoplastea PX-proteins as putative targets for a rational design of anti-parasitic drugs.

Niemann–Pick C1 (NPC1) and NPC2 proteins are indispensable for the export of LDL-derived cholesterol from late endosomes. Mutations in these proteins result in Niemann–Pick type C disease, a lysosomal storage disease. Despite recent reports of the NPC1 structure depicting its overall architecture, the function of its C-terminal luminal domain (CTD) remains poorly understood even though 45% of NPC disease-causing mutations are in this domain. Here, we report a crystal structure at 3.3 Å resolution of NPC1* (residues 314–1,278), which—in contrast to previous lower resolution structures—features the entire CTD well resolved. Notably, all eight cysteines of the CTD form four disulfide bonds, one of which (C909–C914) enforces a specific loop that in turn mediates an interaction with a loop of the N-terminal domain (NTD). Importantly, this loop and its interaction with the NTD were not observed in any previous structures due to the lower resolution. Our mutagenesis experiments highlight the physiological relevance of the CTD–NTD interaction, which might function to keep the NTD in the proper orientation for receiving cholesterol from NPC2. Additionally, this structure allows us to more precisely map all of the disease-causing mutations, allowing future molecular insights into the pathogenesis of NPC disease.

The pathogenesis ofKlebsiella pneumoniais linked to the bacteria’s ability to form biofilms. Mannose-resistantKlebsiella-like (Mrk) hemagglutinins are critical forK.pneumoniabiofilm development, and the expression of the genes encoding these proteins is activated by a 3′,5′-cyclic diguanylic acid (c-di-GMP)–regulated transcription factor, MrkH. To gain insight into MrkH function, we performed structural and biochemical analyses. Data revealed MrkH to be a monomer with a two-domain architecture consisting of a PilZ C-domain connected to an N domain that unexpectedly also harbors a PilZ-like fold. Comparison of apo- and c-di-GMP–bound MrkH structures reveals a large 138° interdomain rotation that is induced by binding an intercalated c-di-GMP dimer. c-di-GMP interacts with PilZ C-domain motifs 1 and 2 (RxxxR and D/NxSxxG) and a newly described c-di-GMP–binding motif in the MrkH N domain. Strikingly, these c-di-GMP–binding motifs also stabilize an open state conformation in apo MrkH via contacts from the PilZ motif 1 to residues in the C-domain motif 2 and the c-di-GMP–binding N-domain motif. Use of the same regions in apo structure stabilization and c-di-GMP interaction allows distinction between the states. Indeed, domain reorientation by c-di-GMP complexation with MrkH, which leads to a highly compacted structure, suggests a mechanism by which the protein is activated to bind DNA. To our knowledge, MrkH represents the first instance of specific DNA binding mediated by PilZ domains. The MrkH structures also pave the way for the rational design of inhibitors that targetK.pneumoniabiofilm formation.

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that utilizes a type III secretion system to subvert host innate immunity. Of the 4 known effector proteins injected into eukaryotic cells, ExoS and ExoU are cytotoxic. The cytotoxic phenotype of ExoU depends on the enzymatic activity of the patatin-like phospholipase A2 domain localized to the N-terminal half of the protein. Amino acid residues located within the C-terminal region of ExoU are postulated to be required for trafficking or localization to the plasma membrane of eukaryotic cells. This report describes the characterization of a transposon-based linker insertion library in ExoU. Utilizing an unbiased screening approach and sensitive methods for measuring enzymatic activity, we identified regions of ExoU that are critical for activation of the phospholipase activity by the only known cofactor, SOD1. Insertions at D572 and L618 reduced the rate of substrate cleavage. Enzymatic activity could be restored to almost parental levels when SOD1 concentrations were increased, suggesting that the linker insertion disrupted the interaction between ExoU and SOD1. An enzyme-linked immunosorbent assay (ELISA)-based binding test was developed to measure ExoU-SOD1 binding. These experiments suggest that ExoU activation by SOD1 is hampered by linker insertion. ExoU derivatives harboring minimal phospholipase activity retained biological activity in tissue culture assays. These proteins affected primarily cellular architecture in a manner similar to that of ExoT. Our studies suggest that conformational changes in ExoU are facilitated by SOD1. Importantly, the level of phospholipase activity influences the biological outcome of ExoU intoxication.

The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNASec). In bacteria, SelA synthesizes Sec from Ser-tRNASec, whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNASec. We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNASec molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNASec-specific D-arm structure, thereby discriminating Ser-tRNASec from Ser-tRNASer. A large cleft is created between two subunits and accommodates the 3′-terminal region of Ser-tRNASec. The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5′-phosphate–dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.

Functional and regulatory features of a protein in a living system are determined by several factors such as chemical nature and three-dimensional arrangement of residues in the functional site, spatial and temporal expression of the protein in the cell and molecules that interact with the protein. Our understanding of molecular determinants of function is never complete without an understanding of the roles of various residues in the protein. All members of a protein family or a protein domain family share a common structural fold and usually have a conserved general function. However, there can be functional specialisation among the members of individual sub-families within the domain families. This specialisation is a consequence of apparently small structural and sequence differences among the members of divergent sub-families within a domain family. In this project, we have classified members of a protein family into sub-families using a rational basis, and then deduced the residues which are specifically conserved within the individual sub-families. The objective of this work is to study the structural and functional specialised roles that the selectively conserved residues are conferring to their respective sub-families. We have considered two protein domain families as case studies for analysis. The first is the SH2 domain family, the members of which have a function of specifically binding to phospho-Tyr residues in proteins. Chapter 2 describes the partitioning of members of the SH2 domain family in the context of the different domain architectures of proteins into which the SH2 domain is incorporated. Two sub-families were defined consisting of proteins with Src-kinase and Socs (suppressor of cytokine signalling) architecture. Domain-architecture specific residues of each subfamily were identified by comparing multiple sequence alignments of SH2 domain sequences from the individual sub-families. Crystal structures of SH2 domains from the Src-kinase and Socs subfamilies were then inspected to determine the structural and functional roles of the selectively conserved residues. Side-chains of many of the selectively conserved residues from each sub-family were found to be involved in functional interactions either with other residues from the non-SH2 regions of the same protein chain or with residues from the phospho-Tyrosine peptide-ligand. This suggests the roles of selectively conserved residues in assisting domain-domain communication within the protein or conferring sequence specificity towards the pTyr-containing sequences to which the SH2 domains from the two sub-families are specialised to bind. The second protein domain family studied is that of the protein kinases. Chapter 3 describes the comparision between two pre-defined sub-families, cyclin-dependent kinase 2 (cdk2) and protein kinase CK2, of Ser/Thr-kinases belonging to the same hierarchical CMGC group. Cdk2-specific and CK2-specific selectively conserved residues were identified through comparison of the respective multiple sequence alignments and their specialised roles were inferred through survey of literature information, including crystal structures of protein-protein complexes involving Cdk2 and CK2. A number of selectively conserved residues from each sub-family were found to be participating in protein-protein interactions involving the respective protein partners of Cdk2 and CK2. Thus, the approach employed in this project helped us to identify subfamily-specific residues which are sites of subfamily-specific functional and/or structural specialisation, and generate diversity within a protein family. Further, the present work enabled us to propose residues which are likely to play roles in conferring subfamily-specific properties and investigation into their roles could form the basis of future experimental studies.

Companies involved in the nuclear energy domain, like component and platform manufacturers, system integrators and utilities, have well established yearly trainings on Nuclear Safety Culture. These trainings are typically covered as part of the annual quality assurance-related refresher trainings, introductory courses for new employees, or indoctrinations of temporary staff. Gradually, security awareness trainings are also addressed on a regular basis, typically with a focus on IT, the daily office work, test bay or construction site work environment, and some data protection and privacy-related topics. Due to emerging national nuclear regulation, steadily but surely, specialized cybersecurity trainings are foreseen for integrators and utilities. Beyond these safety, physical security and cybersecurity specific trainings, there is a need to address the joint part of these disciplines, starting from the planning phase of a new Nuclear Power Plant (NPP). The engineers working on safety, physical protection and cybersecurity, must be aware of these interrelations to jointly elaborate a robust I&C architecture (defense-in-depth, design basis events, functional categorization and systems classification) and a resilient security architecture (security by design, security grading, zone model or infrastructure domain, security conduits, forensic readiness, Security Information and Event Management). This paper provides more in-depth justification of when and where additional training is needed, due to the ubiquitous deployment of digital technology in new NPPs. Additionally, for existing NPPs, the benefits of conveying knowledge by training on specific interfaces between the involved disciplines, will be discussed. Furthermore, the paper will address the need of focused training of management stakeholders, as eventually, they must agree on the residual risk. The decision-makers are in charge of facilitating the inter-disciplinary cooperation in parallel to the allocation of resources, e.g. on security certifications of products, extended modeling-based safety and security analyses and security testing coverage.