Nina M. Haste

Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism

The surface comparison of different serine–threonine and tyrosine kinases reveals a set of 30 residues whose spatial positions are highly conserved. The comparison between active and inactive conformations identified the residues whose positions are the most sensitive to activation. Based on these results, we propose a model of protein kinase activation. This model explains how the presence of a phosphate group in the activation loop determines the position of the catalytically important aspartate in the Asp-Phe-Gly motif. According to the model, the most important feature of the activation is a “spine” formation that is dynamically assembled in all active kinases. The spine is comprised of four hydrophobic residues that we detected in a set of 23 eukaryotic and prokaryotic kinases. It spans the molecule and plays a coordinating role in activated kinases. The spine is disordered in the inactive kinases and can explain how stabilization of the whole molecule is achieved upon phosphorylation.

Nuclease Expression by Staphylococcus aureus Facilitates Escape from Neutrophil Extracellular Traps

Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.

The hallmark of AGC kinase functional divergence is its C-terminal tail, a cis-acting regulatory module

The catalytic activities of eukaryotic protein kinases (EPKs) are regulated by movement of the C-helix, movement of the N and C lobes upon ATP binding, and movement of the activation loop upon phosphorylation. Statistical analysis of the selective constraints associated with AGC kinase functional divergence reveals conserved interactions between these regulatory regions and three regions of the C-terminal tail (C-tail): the N-lobe tether (NLT), the active-site tether (AST), and the C-lobe tether (CLT). The NLT serves as a docking site for an upstream kinase PDK1 and, upon activation, positions the C-helix within the ATP binding pocket. The AST directly interacts with the ATP binding pocket, and the CLT interacts with the interlobe linker and the αC–β4 loop, which appears to serve as a hinge for C-helix movement. The C-tail is a hallmark of AGC functional divergence inasmuch as most of the conserved core residues that distinguish AGC kinases from other EPKs are associated with the NLT, AST, or CLT. Moreover, several AGC catalytic core conserved residues that interact with the C-tail strikingly diverge from the canonical residues observed at corresponding positions in nearly all other EPKs, suggesting that the catalytic core may have coevolved with the C-tail in AGC kinases. These observations, along with the fact that the C-tail is needed for catalytic activity suggests that the C-tail is a cis-acting regulatory module that can also serve as a regulatory “handle,” to which trans-acting cellular components can bind to modulate activity.

Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis

During bacterial cannibalism, a differentiated subpopulation harvests nutrients from their genetically identical siblings to allow continued growth in nutrient-limited conditions. Hypothesis-driven imaging mass spectrometry (IMS) was used to identify metabolites active in a Bacillus subtilis cannibalism system in which sporulating cells lyse nonsporulating siblings. Two candidate molecules with sequences matching the products of skfA and sdpC, genes for the proposed cannibalistic factors sporulation killing factor (SKF) and sporulation delaying protein (SDP), respectively, were identified and the structures of the final products elucidated. SKF is a cyclic 26-amino acid (aa) peptide that is posttranslationally modified with one disulfide and one cysteine thioether bridged to the α-position of a methionine, a posttranslational modification not previously described in biology. SDP is a 42-residue peptide with one disulfide bridge. In spot test assays on solid medium, overproduced SKF and SDP enact a cannibalistic killing effect with SDP having higher potency. However, only purified SDP affected B. subtilis cells in liquid media in fluorescence microscopy and growth assays. Specifically, SDP treatment delayed growth in a concentration-dependent manner, caused increases in cell permeability, and ultimately caused cell lysis accompanied by the production of membrane tubules and spheres. Similarly, SDP but not SKF was able to inhibit the growth of the pathogens Staphylococcus aureus and Staphylococcus epidermidis with comparable IC50 to vancomycin. This investigation, with the identification of SKF and SDP structures, highlights the strength of IMS in investigations of metabolic exchange of microbial colonies and also demonstrates IMS as a promising approach to discover novel biologically active molecules.

PKR and eIF2α: Integration of Kinase Dimerization, Activation, and Substrate Docking

The antiviral RNA-dependent protein kinase, PKR, binds to viral double-stranded RNA in the cell and halts protein synthesis by phosphorylating the alpha subunit of the translation initiation factor eIF2. In this issue of Cell, two complementary papers Dar et al. (2005) and Dey et al. (2005) address the interaction between PKR and eIF2alpha. The structures of eIF2alpha bound to PKR reveal that PKR forms a dimer, the interface of which is essential for kinase activation, and demonstrate how this protein substrate docks to its kinase. The structures, coupled with mutagenesis analysis, also demonstrate how phosphorylation of the activation loop can allosterically couple two distal regions, the dimerization and substrate recognition interfaces.

Related Protein-Protein Interaction Modules Present Drastically Different Surface Topographies Despite A Conserved Helical Platform

The subcellular localization of cAMP-dependent protein kinase (PKA) occurs through interaction with A-Kinase Anchoring Proteins (AKAPs). AKAPs bind to the PKA regulatory subunit dimer of both type Ialpha and type IIalpha (RIalpha and RIIalpha). RIalpha and RIIalpha display characteristic localization within different cell types, which is maintained by interaction of AKAPs with the N-terminal dimerization and docking domain (D/D) of the respective regulatory subunit. Previously, we reported the solution structure of RIIa D/D module, both free and bound to AKAPs. We have now solved the solution structure of the dimerization and docking domain of the type Ialpha regulatory dimer subunit (RIalpha D/D). RIalpha D/D is a compact docking module, with unusual interchain disulfide bonds that help maintain the AKAP interaction surface. In contrast to the shallow hydrophobic groove for AKAP binding across the surface of the RIIalpha D/D dimeric interface, the RIalpha D/D module presents a deep cleft for proposed AKAP binding. RIalpha and RIIalpha D/D interaction modules present drastically differing dimeric topographies, despite a conserved X-type four-helix bundle structure.

Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry

Microbial competition exists in the general environment, such as soil or aquatic habitats, upon or within unicellular or multicellular eukaryotic life forms. The molecular actions that govern microbial competition, leading to niche establishment and microbial monopolization, remain undetermined. The emerging technology of imaging mass spectrometry (IMS) enabled the observation that there is directionality in the metabolic output of the organism Bacillus subtilis when co-cultured with Staphylococcus aureus. The directionally released antibiotic alters S. aureus virulence factor production and colonization. Therefore, IMS provides insight into the largely hidden nature of competitive microbial encounters and niche establishment, and provides a paradigm for future antibiotic discovery.

Protein kinase resource: An integrated environment for phosphorylation research

The protein kinase superfamily is an important group of enzymes controlling cellular signaling cascades. The increasing amount of available experimental data provides a foundation for deeper understanding of details of signaling systems and the underlying cellular processes. Here, we describe the Protein Kinase Resource, an integrated online service that provides access to information relevant to cell signaling and enables kinase researchers to visualize and analyze the data directly in an online environment. The data set is synchronized with Uniprot and Protein Data Bank (PDB) databases and is regularly updated and verified. Additional annotation includes interactive display of domain composition, cross‐references between orthologs and functional mapping to OMIM records. The Protein Kinase Resource provides an integrated view of the protein kinase superfamily by linking data with their visual representation. Thus, human kinases can be mapped onto the human kinome tree via an interactive display. Sequence and structure data can be easily displayed using applications developed for the PKR and integrated with the website and the underlying database. Advanced search mechanisms, such as multiparameter lookup, sequence pattern, and blast search, enable fast access to the desired information, while statistics tools provide the ability to analyze the relationships among the kinases under study. The integration of data presentation and visualization implemented in the Protein Kinase Resource can be adapted by other online providers of scientific data and should become an effective way to access available experimental information. Proteins 2006.

Activity of the streptogramin antibiotic etamycin against methicillin-resistant Staphylococcus aureus.

The alarming rise of hospital- and community-associated methicillin-resistant Staphylococcus aureus (HA- and CA-MRSA) infections has prompted a desperate search for novel antibiotics. We discovered the streptogramin etamycin produced by an actinomycete species isolated from the coast of Fiji, the first time this antibiotic has been identified from a marine microbe. Etamycin was extracted and purified from this strain (CNS-575) and identified as a three-rotamer species by 2D NMR spectroscopy. Etamycin demonstrated potent activity against HA- and CA-MRSA in microbroth dilution assays, with minimum inhibitory concentrations (MIC) as low as 1–2 mg l−1 against HA- and CA-MRSA strains. Furthermore, etamycin was also active against other Gram-positive and several Gram-negative pathogens and was found to be non-cytotoxic at concentrations more than 20-fold above MIC. Etamycin displayed favorable time-kill kinetics compared with the first-line MRSA antibiotic, vancomycin, and also conferred significant protection from mortality in a murine model of systemic lethal MRSA infection. These data emphasize the utility of the marine environment as a relatively untapped source of antibiotics against major drug-resistant human pathogens. These studies will also guide future isolation and preclinical development of depsipeptide anti-MRSA compounds from marine-derived actinomycetes.

Pharmacological Properties of the Marine Natural Product Marinopyrrole A against Methicillin-Resistant Staphylococcus aureus

ABSTRACT The ongoing spread of methicillin-resistant Staphylococcus aureus (MRSA) strains in hospital and community settings presents a great challenge to public health and illustrates the urgency of discovering new antibiotics. Marinopyrrole A is a member of a structurally novel class of compounds identified from a species of marine-derived streptomycetes with evidence of antistaphylococcal activity. We show that marinopyrrole A has potent concentration-dependent bactericidal activity against clinically relevant hospital- and community-acquired MRSA strains, a prolonged postantibiotic effect superior to that of the current first-line agents vancomycin and linezolid, and a favorable resistance profile. Marinopyrrole A showed limited toxicity to mammalian cell lines (at >20× MIC). However, its antibiotic activity against MRSA was effectively neutralized by 20% human serum. A variety of marinopyrrole analogs were isolated from culture or synthetically produced to try to overcome the inhibitory effect of serum. While many of these compounds retained potent bactericidal effect against MRSA, their activities were also inhibited by serum. Marinopyrrole A has significant affinity for plastic and may therefore have potential as a potent anti-MRSA agent in cutaneous, intracatheter, or antibiotic-lock applications.