Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.


Abstract

Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature. Study holds ProTherm entries: 14882, 14883, 14884, 14885, 14886, 14887, 14888, 14889, 14890, 14891, 14892, 14893, 14894, 14895, 14896, 14897, 14898, 14899, 14900, 14901, 14902, 14903, 14904, 14905, 14906, 14908, 14909, 14910, 14911, 14912, 14913, 14914, 14915, 14916 Extra Details: Tm in the presence of approximately 1 mol of Ca2+/mol of protein calcium binding; binding affinity; unfolding temperature

Submission Details

ID: FPq3iS6B4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Abou-Hachem M;Karlsson EN;Simpson PJ;Linse S;Sellers P;Williamson MP;Jamieson SJ;Gilbert HJ;Bolam DN;Holst O,Biochemistry (2002) Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus. PMID:11980476
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