Vogel, B. | Search bioRxiv

bioRxiv2021

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative {beta}7-{beta}8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1 position of the substrate motif, underlined in LPXTG, in contrast to the strict P1 Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of {beta}7-{beta}8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified {beta}7-{beta}8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. We argue that a greater understanding of individual SrtA sequence and structural determinants of target selectivity can facilitate the design or discovery of improved sortagging tools.

biochemistry↗

bioRxiv2021

Large positive ecological changes of small urban greening actions

The detrimental effects of human-induced environmental change on people and other species are acutely manifested in urban environments. While urban greenspaces are known to mitigate these effects and support functionally diverse ecological communities, evidence of the ecological outcomes of urban greening remains scarce. We use a longitudinal observational design to provide empirical evidence of the putative ecological benefits of greening actions. We show how a small greening action quickly led to large positive changes in the richness, demographic dynamics, and network structure of a depauperate insect community. An increase in the diversity and complexity of the plant community led to, after only three years, a large increase in insect species richness, a greater probability of occurrence of insects within the greenspace, and a higher number and diversity of interactions between insects and plant species. We demonstrate how large ecological benefits may be derived from investing in small greening actions and how these contribute to bring indigenous species back to greenspaces where they have become rare or locally extinct. Our findings provide crucial evidence that support best practice in greenspace design and contribute to re-invigorate policies aimed at mitigating the negative impacts of urbanisation on people and other species.

ecology↗