Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein-protein interactions

被引:50
作者
Haldimann, A
Prahalad, MK
Fisher, SL
Kim, SK
Walsh, CT
Wanner, BL
机构
[1] PURDUE UNIV, DEPT BIOL SCI, W LAFAYETTE, IN 47907 USA
[2] HARVARD UNIV, SCH MED, DEPT BIOL CHEM & MOL PHARMACOL, BOSTON, MA 02115 USA
关键词
P1-Int-Xis cloning; protein phosphorylation; two-component regulatory systems; vancomycin resistance;
D O I
10.1073/pnas.93.25.14361
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Two-component regulatory systems require highly specific interactions between histidine kinase (transmitter) and response regulator (receiver) proteins, We have developed a novel genetic strategy that is based on tightly regulated synthesis of a given protein to identify domains and residues of an interacting protein that are critical for interactions between them, Using a reporter strain synthesizing the nonpartner kinase VanS under tight arabinose control and carrying a promoter-lacZ fusion activated by phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB showing enhanced activation (phosphorylation) by VanS as arabinose-dependent Lac(+) mutants. Changes in the PhoB(AR) mutants cluster in a ''patch'' near the proposed helix 4 of PhoB based on the CheY crystal structure (a homolog of the PhoB receiver domain) providing further evidence that helix 4 lies in the kinase-regulator interface, Based on the CheY structure, one mutant has an additional change in a region that mag propagate a conformational change to helix 4. The overall genetic strategy described here may also be useful for studying interactions of other components of the vancomycin resistance and P-i signal transduction pathways, other two-component regulatory systems, and other interacting proteins, Conditionally replicative oriR(R6K gamma) attP ''genome targeting'' suicide plasmids carrying mutagenized phoB coding regions were integrated into the chromosome of a reporter strain to create mutant libraries; plasmids encoding mutant PhoB proteins were subsequently retrieved by P1-Int-Xis cloning. Finally, the use of similar genome targeting plasmids and P1-Int-Xis cloning should be generally useful for constructing genomic libraries from a wide array of organisms.
引用
收藏
页码:14361 / 14366
页数:6
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