Study of the structure and dynamics of a chimeric variant of the SH3 domain (SHA-Bergerac) by NMR spectroscopy

Prokhorov, Dmitry; Timchenko, Maria; Kudrevatykh, Yu. A.; Fedyukina, Daria V.; Gushchina, Liubov V.; Khristoforov, V. S.; Filimonov, Vladimir V.; Kutyshenko, V. P.

doi:10.1134/s1068162008050075

Cited by 4 publications

(1 citation statement)

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“…Yet, the majority of the structural studies of spectrin are of its structural domains (e.g., 1AJ3 43 ; 2SPC 44 ; 3EDV 45 ; 3KBT/ 3KBU 32 ) or SH3 domain (2RMO 46 ; 2OAW 47 ; 1U06 48 ). For the tetramerization regions, many studies have focused on the N-terminal region of aI-and aII-spectrin, [15][16][17][18]30,31,42,48 but only a few have studied the Cterminal region of b-spectrin.…”

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confidence: 99%

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

et al. 2011

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We have screened a human immunoglobulin single-chain variable fragment (scFv) phage library against the C-terminal tetramerization regions of erythroid and nonerythroid beta spectrin (bI-C1 and bII-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid b-spectrin isoforms. We have identified interacting scFvs, with clones ''G5'' and ''A2'' binding only to bI-C1, and clone ''F11'' binding only to bII-C1. The K d values, estimated by competitive enzyme-linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1-0.3 lM. A more quantitative K d value from isothermal titration calorimetry experiments with the recombinant G5 and bI-C1 was 0.15 lM. The a-spectrin fragments (model proteins), aI-N1 and aII-N1, competed with the bI-C1, or bII-C1, binding scFvs, with inhibitory concentration (IC 50 ) values of~50 lM for aI-N1, and 0.5 lM for aII-N1. Our predicted structures of bI-C1 and bII-C1 suggest that the Helix B 0 of the Cterminal partial domain of bI differs from that of bII. Consequently, an unstructured region downstream of Helix B 0 in bI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in bII was helical, and bII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different b-spectrin isoforms to regulate a-and b-spectrin association to form functional spectrin tetramers, and may sort b-spectrin isoforms to their specific cellular localizations.Keywords: beta spectrin; erythroid and nonerythroid; scFv; structural difference; affinity difference Abbreviations: aI-N1, a recombinant protein of aI-spectrin segment with residues 1-156; aI-spectrin, erythroid a-spectrin; aII-N1, a GST fusion protein of aII-spectrin segment with residues 1-147; aII-spectrin, nonerythroid a-spectrin; bI-C1, a GST fusion protein of bI-spectrin segment with residues 1898-2083; bI-C1D, a GST fusion protein of bI-spectrin segment with residues 1898-2070, that is, the residues 2071-2083 in bI-C1 were deleted; bI-spectrin, erythroid b-spectrin; bII-C1, a GST fusion protein of bII-spectrin segment with residues 1906-2093; bII-spectrin, nonerythroid b-spectrin; CD, circular dichroism; CDRs, complementarity determining regions; ELISA, enzyme-linked immunosorbent assay; EPR, electron paramagnetic resonance; GST, glutathione S-transferase; HRP, horseradish peroxidase; ITC, isothermal titration calorimetry; PBS, phosphate buffered saline at pH 7.4; rG5, recombinant G5, a single-chain variable fragment found to bind bI-C1; scFv, single-chain variable fragment.Yuanli Song's current address is

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