UCSF

Antibiotic use has led to the evolution of antibiotic resistance enzymes able to hydrolyze newer antibiotics, leaving them useless against killing bacteria. Are there biophysical costs to evolving drug resistance? The hypothesis addressed here is that both protein stability and native activity constrain enzyme evolution, and uses the enzyme beta-lactamase, the major resistance mechanism against beta-lactam antibiotics like penicillin, to investigate this question. Beta-lactamase hydrolyzes beta-lactam antibiotics, such as penicillin, rendering them ineffective. In response to drugs combating beta-lactamase, two types of beta-lactamase resistance mutants have emerged in the clinic. Extended-spectrum mutants have gained the ability to break down larger, third-generation antibiotics that were previously impervious to beta-lactamase, whereas inhibitor-resistant mutants have decreased inhibition by covalent inhibitors. Each category of resistance mutant was investigated. First, the crystallographic structure of the S130G inhibitor-resistant beta-lactamase was determined to 1.4 Ångstrom resolution. Two configurations of a new water molecule were observed in the active site, and explain how this resistance mutant remains active with the presumed catalytic acid for substrate hydrolysis replaced. Substituting this key catalytic residue to gain inhibitor resistance resulted in a mutant enzyme with almost ten-thousand-fold decreased catalytic activity -- a rather Pyrrhic victory, and an example of the biophysical cost enzymes pay for developing drug resistance.

Second, the "stability-function" hypothesis - do drug-resistant mutants, and enzymes in general, sacrifice their inherent stability and ancestral activity as they gain activity against newer antibiotics or substrates - was investigated in beta-lactamase extended-spectrum resistance mutants. The thermodynamic stability and catalytic activity against both third-generation and older antibiotics was determined for five known beta-lactamase extended-spectrum mutant enzymes. These mutant enzymes had between 100- to 200-fold increased activity against the antibiotic cefotaxime, but were destabilized by up to 4.1 kcal/mol, consistent with the stability-function hypothesis. Eight structures, including complexes with inhibitors and extended-spectrum antibiotics, were determined by x-ray crystallography. Distinct mechanisms of action were revealed for each mutant, including changes in the flexibility and ground state structures of the enzyme. Understanding biophysical constraints on evolution of drug resistance, and how distant substitutions are structurally transmitted to the active site has broad relevance to drug-resistant systems.