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Antibiotic resistance in clinically important bacteria can be mediated by target protection mechanisms, whereby a protein binds to the drug target and protects it from the inhibitory effects of the antibiotic. The most prevalent source of clinical resistance to the antibiotic fusidic acid (FA) is expression of the FusB family of proteins that bind to the drug target (Elongation factor G [EF-G]) and promote dissociation of EF-G from FA-stalled ribosome complexes. FusB binding causes changes in both the structure and conformational flexibility of EF-G, but which of these changes drives FA resistance was not understood. We present here detailed characterization of changes in the conformational flexibility of EF-G in response to FusB binding and show that these changes are responsible for conferring FA resistance. Binding of FusB to EF-G causes a significant change in the dynamics of domain III of EF-G C3 that leads to an increase in a minor, more disordered state of EF-G domain III. This is sufficient to overcome the steric block of transmission of conformational changes within EF-G by which FA prevents release of EF-G from the ribosome. This study has identified an antibiotic resistance mechanism mediated by allosteric effects on the dynamics of the drug target., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Antibiotic resistance in clinically important bacteria can be mediated by proteins that physically associate with the drug target and act to protect it from the inhibitory effects of an antibiotic. We present here the first detailed structural characterization of such a target protection mechanism mediated through a protein-protein interaction, revealing the architecture of the complex formed between the FusB fusidic acid resistance protein and the drug target (EF-G) it acts to protect. Binding of FusB to EF-G induces conformational and dynamic changes in the latter, shedding light on the molecular mechanism of fusidic acid resistance.

Temperature coefficients have been measured for backbone amide (1)H and (15)N nuclei in the B1 domain of protein G (GB1), using temperatures in the range 283-313 K, and pH values from 2.0 to 9.0. Many nuclei display pH-dependent coefficients, which were fitted to one or two pK(a) values. (1)H coefficients showed the expected behaviour, in that hydrogen-bonded amides have less negative values, but for those amides involved in strong hydrogen bonds in regular secondary structure there is a negative correlation between strength of hydrogen bond and size of temperature coefficient. The best correlation to temperature coefficient is with secondary shift, indicative of a very approximately uniform thermal expansion. The largest pH-dependent changes in coefficient are for amides in loops adjacent to sidechain hydrogen bonds rather than the amides involved directly in hydrogen bonds, indicating that the biggest determinant of the temperature coefficient is temperature-dependent loss of structure, not hydrogen bonding. Amide (15)N coefficients have no clear relationship with structure.

A number of signals in the NMR spectrum of the B1 domain of staphylococcal protein G (GB1) show a chemical shift dependence on the concentration of the protein at pH 3 but not at neutral pH, implying the existence of self-association at low pH. NMR backbone relaxation experiments show that GB1 undergoes a slow conformational exchange at pH 3, which is not seen at higher pH. Analysis of relaxation dispersion experiments yields a self-association constant of 50 mM, and shows that (15)N chemical shift changes in the dimer interface are up to 3 ppm. The shift changes measured from concentration-dependent HSQC spectra and from relaxation dispersion show good consistency. Measurements of chemical shifts as a function of pH show that a hydrogen bond between the sidechains of Asp44 and Gln40 is broken when Asp44 is protonated, and that loss of this hydrogen bond leads to the breaking of the (i, i + 4) backbone helical hydrogen bond from Asp44 HN to Gln40 O, and therefore to a loss of two residues from the C-terminal end of the helix. This weakens the helix structure and facilitates the loss of further helical structure thus permitting dimerization, which is suggested to occur in the same way as observed for the A42F mutant of GB1 (Jee et al., Proteins 2007;71:1420-1431), by formation of an antiparallel beta-sheet between the edge strands 2 in two monomers. The monomer/dimer ratio is thus a finely balanced equilibrium even in the wild type protein., (Proteins 2010. (c) 2009 Wiley-Liss, Inc.)

NMR investigations have been carried out on the B1 domain of protein G. This protein has six lysine residues, of which three are consistently found to form surface-exposed salt bridges in crystal structures, while the other three are not. The Nzeta and Hzeta chemical shifts of all six lysines are similar and are not affected significantly by pH titration of the carboxylate groups in the protein, except for a relatively small titration of K39 Nzeta. Deuterium isotope effects on nitrogen and proton are of the size expected for a simple hydrated amine (a result supported by density functional theory calculations), and also do not titrate with the carboxylates. The line shapes of the J-coupled (15)N signals suggest rapid internal reorientation of all NH(3)(+) groups. pK(a) values have been measured for all charged side chains except Glu50 and do not show the perturbations expected for salt bridge formation, except that E35 has a Hill coefficient of 0.84. The main differential effect seen is that the lysines that are involved in salt bridges in the crystal display faster exchange of the amine protons with the solvent, an effect attributed to general base catalysis by the carboxylates. This explanation is supported by varying buffer composition, which demonstrates reduced electrostatic shielding at low concentration. In conclusion, the study demonstrates that the six surface-exposed lysines in protein G are not involved in significant salt bridge interactions, even though such interactions are found consistently in crystal structures. However, the intrahelical E35-K39 (i,i+4) interaction is partially present.