Crystal Structure of Staphopain C from Staphylococcus aureus.

Staphylococcus aureus is a common opportunistic pathogen of humans and livestock that causes a wide variety of infections. The success of S. aureus as a pathogen depends on the production of an array of virulence factors including cysteine proteases (staphopains)-major secreted proteases of certain strains of the bacterium. Here, we report the three-dimensional structure of staphopain C (ScpA2) of S. aureus, which shows the typical papain-like fold and uncovers a detailed molecular description of the active site. Because the protein is involved in the pathogenesis of a chicken disease, our work provides the foundation for inhibitor design and potential antimicrobial strategies against this pathogen.

Crystal Structure of Mannose Specific IIA Subunit of Phosphotransferase System from Streptococcus pneumoniae.

Streptococcus pneumoniae is a frequent bacterial pathogen of the human respiratory tract causing pneumonia, meningitis and sepsis, a serious healthcare burden in all age groups. S. pneumoniae lacks complete respiratory chain and relies on carbohydrate fermentation for energy generation. One of the essential components for this includes the mannose phosphotransferase system (Man-PTS), which plays a central role in glucose transport and exhibits a broad specificity for a range of hexoses. Importantly, Man-PTS is involved in the global regulation of gene expression for virulence determinants. We herein report the three-dimensional structure of the EIIA domain of S. pneumoniae mannose phosphotransferase system (SpEIIA-Man). Our structure shows a dimeric arrangement of EIIA and reveals a detailed molecular description of the active site. Since PTS transporters are exclusively present in microbes and sugar transporters have already been suggested as valid targets for antistreptococcal antibiotics, our work sets foundation for the future development of antimicrobial strategies against Streptococcus pneumoniae.

Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)-A CleavEx Based Analysis.

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.

Development of Chemical Tools to Monitor Human Kallikrein 13 (KLK13) Activity.

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.

An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.

BACKGROUND: Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential. METHODOLOGY/PRINCIPAL FINDINGS: Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H(+)-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials. CONCLUSIONS/SIGNIFICANCE: Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.