Using cellular fitness to map the structure and function of a major facilitator superfamily effluxer.

The major facilitator superfamily (MFS) effluxers are prominent mediators of antimicrobial resistance. The biochemical characterization of MFS proteins is hindered by their complex membrane environment that makes in vitro biochemical analysis challenging. Since the physicochemical properties of proteins drive the fitness of an organism, we posed the question of whether we could reverse that relationship and derive meaningful biochemical parameters for a single protein simply from fitness changes it confers under varying strengths of selection. Here, we present a physiological model that uses cellular fitness as a proxy to predict the biochemical properties of the MFS tetracycline efflux pump, TetB, and a family of single amino acid variants. We determined two lumped biochemical parameters roughly describing Km and Vmax for TetB and variants. Including in vivo protein levels into our model allowed for more specified prediction of pump parameters relating to substrate binding affinity and pumping efficiency for TetB and variants. We further demonstrated the general utility of our model by solely using fitness to assay a library of tet(B) variants and estimate their biochemical properties

Small changes in enzyme function can lead to surprisingly large fitness effects during adaptive evolution of antibiotic resistance

In principle, evolutionary outcomes could be largely predicted if all of the relevant physicochemical variants of a particular protein function under selection were known and integrated into an appropriate physiological model. We have tested this principle by generating a family of variants of the tetracycline resistance protein TetX2 and identified the physicochemical properties most correlated with organismal fitness. Surprisingly, small changes in the Km(MCN), less than twofold, were sufficient to produce highly successful adaptive mutants over clinically relevant drug concentrations. We then built a quantitative model directly relating the in vitro physicochemical properties of the mutant enzymes to the growth rates of bacteria carrying a single chromosomal copy of the tet(X2) variants over a wide range of minocycline (MCN) concentrations. Importantly, this model allows the prediction of enzymatic properties directly from cellular growth rates as well as the physicochemical-fitness landscape of TetX2. Using experimental evolution and deep sequencing to monitor the allelic frequencies of the seven most biochemically efficient TetX2 mutants in 10 independently evolving populations, we showed that the model correctly predicted the success of the two most beneficial variants tet(X2)T280A and tet(X2)N371I. The structure of the most efficient variant, TetX2T280A, in complex with MCN at 2.7 Å resolution suggests an indirect effect on enzyme kinetics. Taken together, these findings support an important role for readily accessible small steps in protein evolution that can, in turn, greatly increase the fitness of an organism during natural selection

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State

The cyclic antimicrobial lipopeptide daptomycin (DAP) triggers the LiaFSR membrane stress response pathway in enterococci and many other Gram-positive organisms. LiaR is the response regulator that, upon phosphorylation, binds in a sequence-specific manner to DNA to regulate transcription in response to membrane stress. In clinical settings, non-susceptibility to DAP by Enterococcus faecium is correlated frequently with a mutation in LiaR of Trp73 to Cys (LiaRW73C). We have determined the structure of the activated E. faecium LiaR protein at 3.2 Å resolution and, in combination with solution studies, show that the activation of LiaR induces the formation of a LiaR dimer that increases LiaR affinity at least 40-fold for the extended regulatory regions upstream of the liaFSR and liaXYZ operons. In vitro, LiaRW73C induces phosphorylation-independent dimerization of LiaR and provides a biochemical basis for non-susceptibility to DAP by the upregulation of the LiaFSR regulon. A comparison of the E. faecalis LiaR, E. faecium LiaR, and the LiaR homolog from Staphylococcus aureus (VraR) and the mutations associated with DAP resistance suggests that physicochemical properties such as oligomerization state and DNA specificity, although tuned to the biology of each organism, share some features that could be targeted for new antimicrobials

Instruction in the Responsible Conduct of Research: An Inventory of Programs and Materials within CTSAs

The National Institutes of Health (NIH) require instruction in the responsible conduct of research (RCR) as a component of any Clinical and Translational Science Award (CTSA). The Educational Materials Group of the NIH CTSA Consortium's Clinical Research Ethics Key Function Committee (CRE-KFC) conducted a survey of the 38 institutions that held CTSA funding as of January 2009 to determine how they satisfy RCR training requirements. An 8-item questionnaire was sent by email to directors of the Clinical Research Ethics, the Educational and Career Development, and the Regulatory Knowledge cores. We received 78 completed surveys from 38 CTSAs (100%). We found that there is no unified approach to RCR training across CTSAs, many programs lack a coherent plan for RCR instruction, and most CTSAs have not developed unique instructional materials tailored to the needs of clinical and translational scientists. We recommend collaboration among CTSAs and across CTSA key function committees to address these weaknesses. We also requested that institutions send electronic copies of original RCR training materials to share among CTSAs via the CTSpedia website. Twenty institutions submitted at least one educational product. The CTSpedia now contains more than 90 RCR resources.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79071/1/j.1752-8062.2010.00193.x.pd

Molecular basis of FIR-mediated c-myc transcriptional control

The far upstream element (FUSE) regulatory system promotes a peak in the concentration of c-Myc during cell cycle. First, the FBP transcriptional activator binds to the FUSE DNA element upstream of the c-myc promoter. Then, FBP recruits its specific repressor (FIR), which acts as an on/off transcriptional switch. Here we describe the molecular basis of FIR recruitment, showing that the tandem RNA recognition motifs of FIR provide a platform for independent FUSE DNA and FBP protein binding and explaining the structural basis of the reversibility of the FBP-FIR interaction. We also show that the physical coupling between FBP and FIR is modulated by a flexible linker positioned sequentially to the recruiting element. Our data explain how the FUSE system precisely regulates c-myc transcription and suggest that a small change in FBP-FIR affinity leads to a substantial effect on c-Myc concentration.MRC Grant-in-aid U11757455

Structure of an archaeal PCNA1-PCNA2-FEN1 complex: elucidating PCNA subunit and client enzyme specificity.

The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1-PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 A resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1-PCNA2-PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme 'clients', and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades