DNA in cells is supercoiled and constrained into loops and this supercoiling and looping influence every aspect of DNA activity. We show here that negative supercoiling transmits mechanical stress along the DNA backbone to disrupt base pairing at specific distant sites. Cooperativity among distant sites localizes certain sequences to superhelical apices. Base pair disruption allows sharp bending at superhelical apices, which facilitates DNA writhing to relieve torsional strain. The coupling of these processes may help prevent extensive denaturation associated with genomic instability. Our results provide a model for how DNA can form short loops, which are required for many essential processes, and how cells may use DNA loops to position nicks to facilitate repair. Furthermore, our results reveal a complex interplay between site-specific disruptions to base pairing and the 3-D conformation of DNA, which influences how genomes are stored, replicated, transcribed, repaired, and many other aspects of DNA activity.

Lys234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser130, it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase-mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, while Lys 234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a five-fold increase in kcat for ampicillin, suggesting the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest the slow acylation by the K234R enzyme is due to a conformational change in Ser130, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance.

CTX-M β-lactamases are prevalent antibiotic resistance enzymes and are notable for their ability to rapidly hydrolyze the extended-spectrum cephalosporin, cefotaxime. We hypothesized that the active site sequence requirements of CTX-M-mediated hydrolysis differ between classes of β-lactam antibiotics. Accordingly, we use codon randomization, antibiotic selection, and deep sequencing to determine the CTX-M active-site residues required for hydrolysis of cefotaxime and the penicillin, ampicillin. The study reveals positions required for hydrolysis of all β-lactams, as well as residues controlling substrate specificity. Further, CTX-M enzymes poorly hydrolyze the extended-spectrum cephalosporin, ceftazidime. We further show that the sequence requirements for ceftazidime hydrolysis follow those of cefotaxime, with the exception that key active-site omega loop residues are not required, and may be detrimental, for ceftazidime hydrolysis. These results provide insights into cephalosporin hydrolysis and demonstrate that changes to the active-site omega loop are likely required for the evolution of CTX-M-mediated ceftazidime resistance.

Abstractβ-lactamases inactivate β-lactam antibiotics leading to drug resistance. Consequently, inhibitors of β-lactamases can combat this resistance, and the β-lactamase inhibitory protein (BLIP) is a naturally occurring inhibitor. The widespread CTX-M-14 and CTX-M-15 β-lactamases have an 83% sequence identity. In this study, we show that BLIP weakly inhibits CTX-M-14 but potently inhibits CTX-M-15. The structure of the BLIP/CTX-M-15 complex reveals that binding is associated with a conformational change of an active site loop of β-lactamase. Surprisingly, the loop structure in the complex is similar to that in a drug-resistant variant (N106S) of CTX-M-14. We hypothesized that the pre-established favorable loop conformation of the N106S mutant would facilitate binding. The N106S substitution results in a ~100- and 10-fold increase in BLIP inhibition potency for CTX-M-14 and CTX-M-15, respectively. Thus, this indicates that an active site loop in β-lactamase toggles between conformations that control antibiotic hydrolysis and inhibitor susceptibility. These findings highlight the role of accessible active site conformations in controlling enzyme activity and inhibitor susceptibility as well as the influence of mutations in selectively stabilizing discrete conformations.

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