Employing directed evolution for the functional analysis of multi-specific proteins.

Multi-specific proteins located at the heart of complex protein-protein interaction (PPI) networks play essential roles in the survival and fitness of the cell. In addition, multi-specific or promiscuous enzymes exhibit activity toward a wide range of substrates so as to increase cell evolvability and robustness. However, despite their high importance, investigating the in vivo function of these proteins is difficult, due to their complex nature. Typically, deletion of these proteins leads to the abolishment of large PPI networks, highlighting the difficulty in examining the contributions of specific interactions/activities to complex biological processes and cell phenotypes. Protein engineering approaches, including directed evolution and computational protein design, allow for the generation of multi-specific proteins in which certain activities remain intact while others are abolished. The generation and examination of these mutants both in vitro and in vivo can provide high-resolution analysis of biological processes and cell phenotypes and provide new insight into the evolution and molecular function of this important protein family.

Covalent inhibition of bacterial quorum sensing.

Chemical coordination of gene expression among bacteria as a function of population density is regulated by a mechanism known as 'quorum sensing' (QS). QS in Pseudomonas aeruginosa, an opportunistic pathogen that causes disease in immunocompromised patients, is mediated by binding of the transcriptional activator, LasR, to its ligand, 3-oxo-C(12)-HSL, leading to population-wide secretion of virulence factors and biofilm formation. We have targeted QS in P. aeruginosa with a set of electrophilic probes designed to covalently bind Cys79 in the LasR binding pocket, leading to specific inhibition of QS-regulated gene expression and concomitant reduction of virulence factor secretion and biofilm formation. This first example of covalent modification of a QS receptor provides a new tool to study molecular mechanisms of bacterial group behavior and could lead to new strategies for targeting bacterial virulence.

The transition of human estrogen sulfotransferase from generalist to specialist using directed enzyme evolution.

Broad specificity is believed to be a property of primordial enzymes that diverged during natural protein evolution to produce highly specific and efficient enzymes. Human estrogen sulfotransferase (SULT1E1) is a broad-specificity enzyme that detoxifies a variety of chemicals, including estrogens, by the transfer of sulfate. To study the molecular basis for the broad specificity of this enzyme and to investigate the process of SULT1E1 specialization, we have adopted a directed enzyme evolution approach. Using two iterative rounds of evolution, we generated SULT1E1 mutants with increased thermostability and narrower specificity from the broadly specific wild-type enzyme. To identify mutants with enhanced specificity, we developed an unbiased screening assay to assess sulfate transfer to three different acceptors in parallel. Such an assay enabled the isolation of SULT1E1 mutants with enhanced or wild-type activity toward an estrogen acceptor and significantly reduced activity for phenol or coumarin type of acceptors, leading to up to 3 orders of magnitude increase in specificity. We found that mutations conferring novel specificity are located in the vicinity of the active site and thus may play a direct role in reshaping the acceptor-binding site. Finally, such mutations resulted in reduced SULT1E1 thermostability, revealing a trade-off between SULT1E1 thermostability and acquisition of novel function.

The molecular basis for the broad substrate specificity of human sulfotransferase 1A1.

Cytosolic sulfotransferases (SULTs) are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.