Structural and Functional Insights Into Lysostaphin-Substrate Interaction.

Lysostaphin from Staphylococcus simulans and its family enzymes rapidly acquire prominence as the next generation agents in treatment of S. aureus infections. The specificity of lysostaphin is promoted by its C-terminal cell wall targeting domain selectivity toward pentaglycine bridges in S. aureus cell wall. Scission of these cross-links is carried out by its N-terminal catalytic domain, a zinc-dependent endopeptidase. Understanding the determinants affecting the efficiency of catalysis and strength and specificity of interactions lies at the heart of all lysostaphin family enzyme applications. To this end, we have used NMR, SAXS and molecular dynamics simulations to characterize lysostaphin structure and dynamics, to address the inter-domain interaction, the enzyme-substrate interaction as well as the catalytic properties of pentaglycine cleavage in solution. Our NMR structure confirms the recent crystal structure, yet, together with the molecular dynamics simulations, emphasizes the dynamic nature of the loops embracing the catalytic site. We found no evidence for inter-domain interaction, but, interestingly, the SAXS data delineate two preferred conformation subpopulations. Catalytic H329 and H360 were observed to bind a second zinc ion, which reduces lysostaphin pentaglycine cleaving activity. Binding of pentaglycine or its lysine derivatives to the targeting domain was found to be of very low affinity. The pentaglycine interaction site was located to the N-terminal groove of the domain. Notably, the targeting domain binds the peptidoglycan stem peptide Ala-d-γ-Glu-Lys-d-Ala-d-Ala with a much higher, micromolar affinity. Binding site mapping reveals two interaction sites of different affinities on the surface of the domain for this peptide.

Identification and structural characterization of LytU, a unique peptidoglycan endopeptidase from the lysostaphin family.

We introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the overall structure common to the lysostaphin family. Purified LytU lyses S. aureus cells and cleaves pentaglycine, a reaction conveniently monitored by NMR spectroscopy. Substituting the cofactor zinc ion with a copper or cobalt ion remarkably increases the rate of pentaglycine cleavage. NMR and isothermal titration calorimetry further reveal that, uniquely for its family, LytU is able to bind a second zinc ion which is coordinated by catalytic histidines and is therefore inhibitory. The pH-dependence and high affinity of binding carry further physiological implications.

1H, 13C and 15N resonance assignments of the new lysostaphin family endopeptidase catalytic domain from Staphylococcus aureus.

Lysostaphin family endopeptidases, produced by Staphylococcus genus, are zinc-dependent enzymes that cleave pentaglycine bridges of cell wall peptidoglycan. They act as autolysins to maintain cell wall metabolism or as toxins and weapons against competing strains. Consequently, these enzymes are compelling targets for new drugs as well as are potential antimicrobial agents themselves against Staphylococcus pathogens, which depend on cell wall to retain their immunity against antibiotics. The rapid spread of methicillin and vancomycin-resistant Staphylococcus aureus strains draws demand for new therapeutic approaches. S. aureus gene sa0205 was found to be implicated in resistance to vancomycin and synthesis of the bacteria cell wall. The gene encodes for a catalytic domain of a lysostaphin-type endopeptidase. We aim to obtain the structure of the Sa0205 catalytic domain, the first solution structure of the catalytic domain of the lysostaphin family enzymes. In addition, we are to investigate the apparent binding of the second zinc ion, which has not been previously reported for the enzyme group. Herein, we present the backbone and side chain resonance assignments of Sa0205 endopeptidase catalytic domain in its one and two zinc-bound forms.

Segmental isotopic labeling of multi-domain and fusion proteins by protein trans-splicing in vivo and in vitro.

Segmental isotopic labeling is a powerful labeling technique for reducing nuclear magnetic resonance (NMR) signal overlap, which is associated with larger proteins by incorporating stable isotopes into only one region of a protein for NMR detections. Segmental isotopic labeling can not only reduce complexities of NMR spectra but also retain possibilities to carry out sequential resonance assignments by triple-resonance NMR experiments. We described in vivo (i.e., in Escherichia coli) and in vitro protocols for segmental isotopic labeling of multi-domain and fusion proteins via protein trans-splicing (PTS) using split DnaE intein without any refolding steps or alpha-thioester modification. The advantage of PTS approach is that it can be carried out in vivo by time-delayed dual-expression system with two controllable promoters. A segmentally isotope-labeled protein can be expressed in Escherichia coli within 1 d once required vectors are constructed. The total preparation time of a segmentally labeled sample can be as short as 7-13 d depending on the protocol used.