TY - JOUR
T1 - NMR characterization of the metallo-β-lactamase from Bacteroides fragilis and its interaction with a tight-binding inhibitor
T2 - Role of an active-site loop
AU - Scrofani, Sergio D.B.
AU - Chung, John
AU - Huntley, James J.A.
AU - Benkovic, Stephen J.
AU - Wright, Peter E.
AU - Dyson, H. Jane
PY - 1999/11/2
Y1 - 1999/11/2
N2 - Understanding the structure and dynamics of the enzymes that mediate antibiotic resistance of pathogenic bacteria will allow us to take steps to combat this increasingly serious public health hazard. Complete backbone NMR resonance assignments have been made for the broad-specificity metallo-β- lactamase CcrA from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. Chemical shift indices show that the secondary structure of the CcrA molecule in solution is very similar to that in published crystal structures. A loop adjacent to the two-zinc catalytic site exhibits significant structural variation in the published structures, but appears from the NMR experiments to be a regular β-hairpin. Backbone heteronuclear NOE measurements indicate that this region has slightly greater flexibility on a picosecond to nanosecond time scale than the molecule as a whole. The loop appears to have an important role in the binding of substrates and inhibitors. Binding of the inhibitor 3-[2'-(S)-benzyl-3'- mercaptopropanoyl]-4-(S)-carboxy-5,5-dimethylthiazolidine causes a marked increase in the stability of the protein toward unfolding and aggregation, and causes changes in the NMR resonance frequencies of residues close to the active (zinc-binding) site, including the β-hairpin loop. There is a small but significant increase in the heteronuclear NOE for this loop upon inhibitor binding, indicative of a decrease in flexibility. In particular, the NOE of the indole ring of tryptophan 49, at the tip of the β-hairpin loop, changes from a low value characteristic of a random coil chain to a significantly higher value, close to that observed for the backbone amides in this region of the protein. These results strongly suggest that the hairpin loop participates in the binding of substrate and in the shielding of the zinc sites from solvent. The broad specificity of the CcrA metallo-β- lactamase may in fact reside in the plasticity of this part of the protein, which allows it to accommodate and bind tightly to substrates of a variety of shapes and sizes.
AB - Understanding the structure and dynamics of the enzymes that mediate antibiotic resistance of pathogenic bacteria will allow us to take steps to combat this increasingly serious public health hazard. Complete backbone NMR resonance assignments have been made for the broad-specificity metallo-β- lactamase CcrA from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. Chemical shift indices show that the secondary structure of the CcrA molecule in solution is very similar to that in published crystal structures. A loop adjacent to the two-zinc catalytic site exhibits significant structural variation in the published structures, but appears from the NMR experiments to be a regular β-hairpin. Backbone heteronuclear NOE measurements indicate that this region has slightly greater flexibility on a picosecond to nanosecond time scale than the molecule as a whole. The loop appears to have an important role in the binding of substrates and inhibitors. Binding of the inhibitor 3-[2'-(S)-benzyl-3'- mercaptopropanoyl]-4-(S)-carboxy-5,5-dimethylthiazolidine causes a marked increase in the stability of the protein toward unfolding and aggregation, and causes changes in the NMR resonance frequencies of residues close to the active (zinc-binding) site, including the β-hairpin loop. There is a small but significant increase in the heteronuclear NOE for this loop upon inhibitor binding, indicative of a decrease in flexibility. In particular, the NOE of the indole ring of tryptophan 49, at the tip of the β-hairpin loop, changes from a low value characteristic of a random coil chain to a significantly higher value, close to that observed for the backbone amides in this region of the protein. These results strongly suggest that the hairpin loop participates in the binding of substrate and in the shielding of the zinc sites from solvent. The broad specificity of the CcrA metallo-β- lactamase may in fact reside in the plasticity of this part of the protein, which allows it to accommodate and bind tightly to substrates of a variety of shapes and sizes.
UR - http://www.scopus.com/inward/record.url?scp=0033517787&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033517787&partnerID=8YFLogxK
U2 - 10.1021/bi990986t
DO - 10.1021/bi990986t
M3 - Article
C2 - 10545172
AN - SCOPUS:0033517787
SN - 0006-2960
VL - 38
SP - 14507
EP - 14514
JO - Biochemistry
JF - Biochemistry
IS - 44
ER -