5XZJ

Crystal structure of the Zn-directed tetramer of the engineered cyt cb562 variant, C96T/AB5

Summary for 5XZJ

• Entry DOI: 10.2210/pdb5xzj/pdb
• Descriptor: Soluble cytochrome b562, HEME C, ZINC ION, ... (5 entities in total)
• Functional Keywords: de novo protein, electron transport
• Biological source: Escherichia coli
• Total number of polymer chains: 4
• Total formula weight: 50705.92

Authors

Song, W.J.,Tezcan, F.A. (deposition date: 2017-07-12, release date: 2017-12-20, Last modification date: 2024-11-13)

Primary citation

Song, W.J.,Yu, J.,Tezcan, F.A.
Importance of Scaffold Flexibility/Rigidity in the Design and Directed Evolution of Artificial Metallo-beta-lactamases.
J. Am. Chem. Soc., 139:16772-16779, 2017

PubMed Abstract: We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn:RIDC1, which was constructed from cytochrome cb building blocks via a metal-templating strategy. Previously, we reported that Zn:RIDC1 could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn:AB3, which in turn displayed catalytic activity for the hydrolysis of activated esters and β-lactam antibiotics. Zn:AB3 was subsequently subjected to directed evolution via an in vivo selection strategy, leading to a variant Zn:AB3 which displayed enzyme-like Michaelis-Menten behavior for ampicillin hydrolysis. A criterion for the evolutionary utility or designability of a new protein structure is its ability to accommodate different active sites. With this in mind, we examined whether Zn:RIDC1 could be tailored with alternative Zn coordination sites that could similarly display evolvable catalytic activities. We report here a detailed structural and functional characterization of new variant Zn:AB5, which houses similar, unsaturated Zn coordination sites to those in Zn:AB3, but in completely different microenvironments. Zn:AB5 displays Michaelis-Menten behavior for ampicillin hydrolysis without any optimization. Yet, the subsequent directed evolution of Zn:AB5 revealed limited catalytic improvement, which we ascribed to the local protein rigidity surrounding the Zn centers and the lack of evolvable loop structures nearby. The relaxation of local rigidity via the elimination of adjacent disulfide linkages led to a considerable structural transformation with a concomitant improvement in β-lactamase activity. Our findings reaffirm previous observations that the delicate balance between protein flexibility and stability is crucial for enzyme design and evolution.

PubMed: 28992705
DOI: 10.1021/jacs.7b08981

Experimental method

X-RAY DIFFRACTION (1.98 Å)