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Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis.

ABSTRACT: Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca(2+) -induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca(2+)-myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on (15) N-labeled recoverin to probe main chain conformational dynamics. (15) N NMR relaxation data suggest that Ca(2+)-free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca(2+) levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca(2+)-dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca(2+) saturated extruded state (R): T ? I ? R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (?(ex) ?? < 1). The final step (I ? R) is much slower (?(ex) ?? > 1). The main chain structure of I is similar in part to the structure of half-saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ? I may transiently increase the exposure of Ca(2+)-binding sites to initiate Ca(2+) binding that drives extrusion of the myristoyl group during I ? R.

SUBMITTER: Xu X

PROVIDER: S-EPMC3092842 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis.

Xu Xianzhong X Ishima Rieko R Ames James B JB

Proteins 20110404 6

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca(2+) -induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca(2+)-myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on (15) N-labeled recoverin to probe main chain conformational dynamics. (15) N NMR relaxation data suggest th ...[more]

PMID: 21465563

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Project description:Human XPA is an essential component in the multienzyme nucleotide excision repair (NER) pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219) was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779-2788, Ikegami et al. (1998) Nat. Struct. Biol. 5, 701-706] and shown to consist of a compact zinc-binding core and a loop-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD. Using this structure, the surface of XPA-MBD which interacts with DNA and a fragment of the largest subunit of replication protein A (RPA70 Delta C327: M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6-4-thymidine-cytidine (64TC)], or with DNA in single- or double-stranded form, indicating that the character of the lesion itself is not the driving force for XPA binding DNA. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70 Delta C327. To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using 15N NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA.

Dataset Information

Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis.

Publications

Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis.

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