Project Details

Description

Regulation of a variety of responses in both eukaryotic and prokaryotic
cells involves families of proteins that function as molecular switches to
turn on and off particular effector functions. The switch between inactive
and active states results from conformational changes that are induced
either by covalent modifications of protein side chains or by binding of
small molecules. In prokaryotic cells, a family of response regulator
proteins composed of homologous switch domains and specific effector
domains, mediate responses to changing environmental conditions. The
regulatory domain is turned on by phosphorylation of an aspartate side
chain and turned off by hydrolysis of the acyl phosphate by an intrinsic
phosphatase activity. Phosphorylation of the regulatory domain occurs via
phosphotransfer from an associated histidine protein kinase. These
phosphotransfer mediated signal transduction systems are widespread
throughout the bacterial kingdom and regulate processes such as cell
motility, differentiation, transport, metabolism, and establishment of
host/pathogen interactions. The proposed research focuses on
structure/function analysis of these phosphorylation-activated switch
domains with the goal of understanding the molecular mechanism of their
action. Specifically, what are the mechanisms of phosphotransfer and
phosphate hydrolysis? What is the nature of the conformational change that
is induced by phosphorylation? And how does the regulatory domain transmit
its effects to the effector domain? The bacterial chemotaxis protein,
CheY, which regulates flagellar rotation, is representative of the
phosphorylation-activated regulatory domains. This 128 amino acid single
domain protein has a classic alpha/beta fold consisting of a central five-
stranded parallel beta sheet flanked on both sides by alpha helices. The
active site, located at the C-terminal edge of the beta sheet is composed
of residues that are highly conserved among the family of bacterial
response regulators. The active site acidic pocket, formed by a cluster of
carboxylate side chains, is the site of phosphorylation and divalent metal
ion binding. Using CheY as a model regulatory domain, the mechanisms of
phosphotransfer and dephosphorylation will be addressed by constructing
site specific mutations, characterizing the
phosphorylating/dephosphorylating activities of these altered proteins
using small molecule phospho-donors, and determining the structures of
relevant mutant proteins by X-ray crystallography. The X-ray structure of
the active conformation of CheY will be approached both by screening for
mutations that stabilize the active form, and by using small molecule
phospho-donors capable of phosphorylating CheY in crystals. As a step
towards determining the mechanism of regulation of effector function via
phosphorylation of the switch domain, structural analysis of two additional
response regulators with attached methylesterase (CheB) and DNA binding
(OmpR) effector domains will be initiated.
StatusFinished
Effective start/end date8/1/926/30/19

Funding

  • National Institutes of Health: $203,212.00
  • National Institutes of Health: $316,334.00
  • National Institutes of Health: $461,529.00
  • National Institutes of Health
  • National Institutes of Health: $343,876.00
  • National Institutes of Health: $335,961.00
  • National Institutes of Health: $200,475.00
  • National Institutes of Health: $299,238.00
  • National Institutes of Health: $340,087.00
  • National Institutes of Health: $290,560.00
  • National Institutes of Health: $336,328.00
  • National Institutes of Health: $173,199.00
  • National Institutes of Health: $472,168.00
  • National Institutes of Health: $183,731.00
  • National Institutes of Health
  • National Institutes of Health: $461,529.00
  • National Institutes of Health: $308,498.00
  • National Institutes of Health: $461,529.00
  • National Institutes of Health
  • National Institutes of Health: $43,572.00
  • National Institutes of Health: $171,534.00
  • National Institutes of Health: $127,204.00
  • National Institutes of Health: $361,506.00
  • National Institutes of Health: $194,727.00
  • National Institutes of Health
  • National Institutes of Health: $329,632.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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