Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
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.
Abou-Hachem
Maher
M
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. maher.abou_hachem@biotek.lu.se
Karlsson
Eva Nordberg
EN
Simpson
Peter J
PJ
Linse
Sara
S
Sellers
Peter
P
Williamson
Michael P
MP
Jamieson
Stuart J
SJ
Gilbert
Harry J
HJ
Bolam
David N
DN
Holst
Olle
O
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D016992:Rhodobacter / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D002118:Calcium