BY-kinases: Protein tyrosine kinases like no other.

BY-kinases (for bacterial tyrosine kinases) constitute a family of protein tyrosine kinases that are highly conserved in the bacterial kingdom and occur most commonly as essential components of multicomponent assemblies responsible for the biosynthesis, polymerization, and export of complex polysaccharides involved in biofilm or capsule formation. BY-kinase function has been attributed to a cyclic process involving formation of an oligomeric species, its disassembly into constituent monomers, and subsequent reassembly, depending on the overall phosphorylation level of a C-terminal cluster of tyrosine residues. However, the relationship of this process to the active/inactive states of the enzyme and the mechanism of its integration into the polysaccharide production machinery remain unclear.

Extended DNA-binding interfaces beyond the canonical SAP domain contribute to the function of replication stress regulator SDE2 at DNA replication forks.

Elevated DNA replication stress causes instability of the DNA replication fork and increased DNA mutations, which underlies tumorigenesis. The DNA replication stress regulator silencing-defective 2 (SDE2) is known to bind to TIMELESS (TIM), a protein of the fork protection complex, and enhances its stability, thereby supporting replisome activity at DNA replication forks. However, the DNA-binding activity of SDE2 is not well defined.

Structural basis for the recognition of the bacterial tyrosine kinase Wzc by its cognate tyrosine phosphatase Wzb.

Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster.

Structural interactions define assembly adapter function of a type II secretion system pseudopilin.

The type IV filament superfamily comprises widespread membrane-associated polymers in prokaryotes. The type II secretion system (T2SS), a virulence pathway in many pathogens, belongs to this superfamily. A knowledge gap in understanding of the T2SS is the molecular role of a small "pseudopilin" protein.

NMR solution structures of Runella slithyformis RNA 2'-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity.

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2'-PO4 to NAD+ yielding RNA 2'-OH and ADP-ribose-1',2'-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD+. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD+-bound states.

Methyl NMR spectroscopy: Measurement of dynamics in viral RNA-directed RNA polymerases.

Measurement of nuclear spin relaxation provides a powerful approach to access information about biomolecular conformational dynamics over several orders of magnitude in timescale. In several cases this knowledge in combination with spatial information from three-dimensional structures yields unique insight into protein stability and the kinetics and thermodynamics of their interactions and function. However, due to intrinsic difficulties in studying large systems using solution state nuclear magnetic resonance (NMR) approaches, until recently these measurements were limited to small-to-medium-sized systems.

Sequential Protein Expression and Capsid Assembly in Cell: Toward the Study of Multiprotein Viral Capsids Using Solid-State Nuclear Magnetic Resonance Techniques.

While solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful technique for studying viral capsids, current studies are limited to capsids formed from single proteins or single polyproteins. The ability to selectively label individual protein components within multiprotein viral capsids and the resulting spectral simplification will facilitate the extension of ssNMR techniques to complex viruses. In vitro capsid assembly by combining individually purified, labeled, and unlabeled components in NMR quantities is not a viable option for most viruses.

Cystoviral RNA-directed RNA polymerases: Regulation of RNA synthesis on multiple time and length scales.

P2, an RNA-directed RNA polymerase (RdRP), is encoded on the largest of the three segments of the double-stranded RNA genome of cystoviruses. P2 performs the dual tasks of replication and transcription de novo on single-stranded RNA templates, and plays a critical role in the viral life-cycle. Work over the last few decades has yielded a wealth of biochemical and structural information on the functional regulation of P2, on its role in the spatiotemporal regulation of RNA synthesis and its variability across the Cystoviridae family.

Structural Basis for the Recognition of Eukaryotic Elongation Factor 2 Kinase by Calmodulin.

Binding of Ca(2+)-loaded calmodulin (CaM) activates eukaryotic elongation factor 2 kinase (eEF-2K) that phosphorylates eEF-2, its only known cellular target, leading to a decrease in global protein synthesis. Here, using an eEF-2K-derived peptide (eEF-2KCBD) that encodes the region necessary for its CaM-mediated activation, we provide a structural basis for their interaction. The striking feature of this association is the absence of Ca(2+) from the CaM C-lobe sites, even under high Ca(2+) conditions.

Methyl Relaxation Measurements Reveal Patterns of Fast Dynamics in a Viral RNA-Directed RNA Polymerase.

Molecular dynamics (MD) simulations combined with biochemical studies have suggested the presence of long-range networks of functionally relevant conformational flexibility on the nanosecond time scale in single-subunit RNA polymerases in many RNA viruses. However, experimental verification of these dynamics at a sufficient level of detail has been lacking. Here we describe the fast, picosecond to nanosecond dynamics of an archetypal viral RNA-directed RNA polymerase (RdRp), the 75 kDa P2 protein from cystovirus ϕ12, using analyses of (1)H-(1)H dipole-dipole cross-correlated relaxation at the methyl positions of Ile (δ1), Leu, Val, and Met residues.

Cystoviral polymerase complex protein P7 uses its acidic C-terminal tail to regulate the RNA-directed RNA polymerase P2.

In bacteriophages of the cystovirus family, the polymerase complex (PX) encodes a 75-kDa RNA-directed RNA polymerase (P2) that transcribes the double-stranded RNA genome. Also a constituent of the PX is the essential protein P7 that, in addition to accelerating PX assembly and facilitating genome packaging, plays a regulatory role in transcription. Deletion of P7 from the PX leads to aberrant plus-strand synthesis suggesting its influence on the transcriptase activity of P2.

Regulatory interactions between a bacterial tyrosine kinase and its cognate phosphatase.

The cyclic process of autophosphorylation of the C-terminal tyrosine cluster (YC) of a bacterial tyrosine kinase and its subsequent dephosphorylation following interactions with a counteracting tyrosine phosphatase regulates diverse physiological processes, including the biosynthesis and export of polysaccharides responsible for the formation of biofilms or virulence-determining capsules. We provide here the first detailed insight into this hitherto uncharacterized regulatory interaction at residue-specific resolution using Escherichia coli Wzc, a canonical bacterial tyrosine kinase, and its opposing tyrosine phosphatase, Wzb. The phosphatase Wzb utilizes a surface distal to the catalytic elements of the kinase, Wzc, to dock onto its catalytic domain (WzcCD).

The assembly mode of the pseudopilus: a hallmark to distinguish a novel secretion system subtype.

In gram-negative bacteria, type II secretion systems assemble a piston-like structure, called pseudopilus, which expels exoproteins out of the cell. The pseudopilus is constituted by a major pseudopilin that when overproduced multimerizes into a long cell surface structure named hyper-pseudopilus. Pseudomonas aeruginosa possesses two type II secretion systems, Xcp and Hxc.

D-Maurocalcine, a pharmacologically inert efficient cell-penetrating peptide analogue.

Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional (1)H NMR structure, and activity of D-maurocalcine.

The XcpV/GspI pseudopilin has a central role in the assembly of a quaternary complex within the T2SS pseudopilus.

Gram-negative bacteria use the sophisticated type II secretion system (T2SS) to secrete a large number of exoproteins into the extracellular environment. Five proteins of the T2SS, the pseudopilins GspG-H-I-J-K, are proposed to assemble into a pseudopilus involved in the extrusion of the substrate through the outer membrane channel. Recent structural data have suggested that the three pseudopilins GspI-J-K are organized in a trimeric complex located at the tip of the GspG-containing pseudopilus.

Structure of the Pseudomonas aeruginosa XcpT pseudopilin, a major component of the type II secretion system.

The bacterial type II protein secretion (T2S) and type IV piliation (T4P) systems share several common features. In particular, it is well established that the T2S system requires the function of a pilus-like structure, called pseudopilus, which is built upon assembly of pilin-like subunits, called pseudopilins. Pilins and pseudopilins have a hydrophobic N-terminal region, which precedes an extended hydrophilic C-terminal region.