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DNA-binding specificity and dimerization of the DNA-binding domain of the PEND protein in the chloroplast envelope membrane.

ABSTRACT: The PEND protein is a DNA-binding protein in the inner envelope membrane of a developing chloroplast, which may anchor chloroplast nucleoids. Here we report the DNA-binding characteristics of the N-terminal basic region plus leucine zipper (bZIP)-like domain of the PEND protein that we call cbZIP domain. The basic region of the cbZIP domain diverges significantly from the basic region of known bZIP proteins that contain a bipartite nuclear localization signal. However, the cbZIP domain has the ability to dimerize in vitro. Selection of binding sites from a random sequence pool indicated that the cbZIP domain preferentially binds to a canonical sequence, TAAGAAGT. The binding site was also confirmed by gel mobility shift analysis using a representative binding site within the chloroplast DNA. These results suggest that the cbZIP domain is a unique DNA-binding domain of the chloroplast protein.

SUBMITTER: Sato N

PROVIDER: S-EPMC55719 | biostudies-literature | 2001 Jun

REPOSITORIES: biostudies-literature

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DNA-binding specificity and dimerization of the DNA-binding domain of the PEND protein in the chloroplast envelope membrane.

Sato N N Ohta N N

Nucleic acids research 20010601 11

The PEND protein is a DNA-binding protein in the inner envelope membrane of a developing chloroplast, which may anchor chloroplast nucleoids. Here we report the DNA-binding characteristics of the N-terminal basic region plus leucine zipper (bZIP)-like domain of the PEND protein that we call cbZIP domain. The basic region of the cbZIP domain diverges significantly from the basic region of known bZIP proteins that contain a bipartite nuclear localization signal. However, the cbZIP domain has the a ...[more]

PMID: 11376142

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Project description:Essentials Membrane-binding GLA domains of coagulation factors are essential for proper clot formation. Factor X (FX) is specific to phosphatidylserine (PS) lipids through unknown atomic-level interactions. Molecular dynamics simulations were used to develop the first membrane-bound model of FX-GLA. PS binding modes of FX-GLA were described, and potential PS-specific binding sites identified. SUMMARY:Background Factor X (FX) binds to cell membranes in a highly phospholipid-dependent manner and, in complex with tissue factor and factor VIIa (FVIIa), initiates the clotting cascade. Experimental information concerning the membrane-bound structure of FX with atomic resolution has remained elusive because of the fluid nature of cellular membranes. FX is known to bind preferentially to phosphatidylserine (PS). Objectives To develop the first membrane-bound model of the FX-GLA domain to PS at atomic level, and to identify PS-specific binding sites of the FX-GLA domain. Methods Molecular dynamics (MD) simulations were performed to develop an atomic-level model for the FX-GLA domain bound to PS bilayers. We utilized a membrane representation with enhanced lipid mobility, termed the highly mobile membrane mimetic (HMMM), permitting spontaneous membrane binding and insertion by FX-GLA in multiple 100-ns simulations. In 14 independent simulations, FX-GLA bound spontaneously to the membrane. The resulting membrane-bound models were converted from HMMM to conventional membrane and simulated for an additional 100 ns. Results The final membrane-bound FX-GLA model allowed for detailed characterization of the orientation, insertion depth and lipid interactions of the domain, providing insight into the molecular basis of its PS specificity. All binding simulations converged to the same configuration despite differing initial orientations. Conclusions Analysis of interactions between residues in FX-GLA and lipid-charged groups allowed for potential PS-specific binding sites to be identified. This new structural and dynamic information provides an additional step towards a full understanding of the role of atomic-level lipid-protein interactions in regulating the critical and complex clotting cascade.

Dataset Information

DNA-binding specificity and dimerization of the DNA-binding domain of the PEND protein in the chloroplast envelope membrane.

Publications

DNA-binding specificity and dimerization of the DNA-binding domain of the PEND protein in the chloroplast envelope membrane.

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