Fine mapping of the sequence requirements for binding of beta-lactamase inhibitory protein (BLIP) to TEM-1 beta-lactamase using a genetic screen for BLIP function.
Beta-lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A beta-lactamases with a wide range of affinities. Alanine-scanning mutagenesis was previously performed to identify the amino acid sequence requirements of BLIP for binding the TEM-1, SME-1, SHV-1, and Bla1 beta-lactamases. Twenty-three BLIP residues that contact TEM-1 beta-lactamase in the structure of the complex were mutated to alanine and assayed for inhibition (K(i)) of beta-lactamase to identify two hotspots of binding energy. These studies have been extended by the development of a genetic screen for BLIP function in Escherichia coli. The bla(TEM-1) gene encoding TEM-1 beta-lactamase was inserted into the E. coli pyrF chromosomal locus. Expression of wild-type BLIP from a plasmid in this strain resulted in a large decrease in ampicillin resistance, while introduction of the same plasmid lacking BLIP had no effect on ampicillin resistance. In addition, it was found that when the BLIP alanine-scanning mutants were tested in the strain, the level of ampicillin resistance was proportional to the K(i) of the BLIP mutant. These results indicate that BLIP function can be monitored by the level of ampicillin resistance of the genetic test strain. Each of the 23 BLIP positions examined by alanine scanning was randomized to create libraries containing all possible substitutions at each position. The genetic screen for BLIP function was used to sort the libraries for active mutants, and DNA sequence analysis of functional BLIP mutants identified the sequences required for binding TEM-1 beta-lactamase. The results indicate the BLIP surface is tolerant of substitutions in that many contact positions can be substituted with other amino acid types and retain wild-type levels of function.
Yuan
Ji
J
Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Huang
Wanzhi
W
Chow
Dar-Chone
DC
Palzkill
Timothy
T
D000409:Alanine; D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism
D001426:Bacterial Proteins; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D000409:Alanine