Protein Crystal Engineering of YpAC-IV Using a Strategy of Excess Charge Reduction
The class IV adenylyl cyclase from Yersinia pestis has been engineered by site-specific mutagenesis to facilitate crystallization at neutral pH. The wild-type enzyme crystallized only below pH 5, consistent with the observation of a carboxyl-carboxylate H bond in a crystal contact in the refined structure 2FJT. On the basis of that unliganded structure at 1.9 A resolution, two different approaches were tested with the goal of producing a higher-pH crystal needed for inhibitor complexation and mechanistic studies. In one approach, Asp 19, which forms the growth-limiting dicarboxyl contact in wild-type triclinic crystals, was modified to Ala and Asn in hopes of relieving the acid-dependence of that crystal form. In the other approach, wild-type residues Met 18, Glu 25, and Asp 55 were (individually) changed to lysine to reduce the protein's excess negative charge in hopes of enabling growth of new, higher-pH forms. These three sites were selected based on their high solvent exposure and lack of intraprotein interactions. The D19A and D19N mutants had reduced solubility and did not crystallize. The other three mutants all crystallized, producing several new forms at neutral pH. One of these forms, with the D55K mutant, enabled a product complex at 0.16 nm resolution, structure 3GHX. This structure shows why the new crystal form required the mutation in order to grow at neutral pH. This approach could be useful in other cases where excess negative charge inhibits the crystallization of low-pI proteins.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 980413
- Report Number(s):
- BNL-93331-2010-JA; TRN: US201015%%1798
- Journal Information:
- Crystal Growth and Design, Vol. 9, Issue 8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
CRYSTALLIZATION
CRYSTALS
CYCLASES
ENGINEERING
ENZYMES
GROWTH
INTERACTIONS
LYSINE
MUTAGENESIS
MUTANTS
MUTATIONS
PROTEINS
REDUCTION
RESIDUES
RESOLUTION
SOLUBILITY
SOLVENTS
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