Structural characterization of the apo form of a calcium binding protein from Entamoeba histolytica by hydrogen exchange and its folding to the holo state.
One of the calcium binding proteins from Entamoeba histolytica (EhCaBP) is a 134 amino acid residue long (M(r) approximately 14.9 kDa) double domain EF-hand protein containing four Ca(2+) binding sites. CD and NMR studies reveal that the Ca(2+)-free form (apo-EhCaBP) exists in a partially collapsed form compared to the Ca(2+)-bound (holo) form, which has an ordered structure (PDB ID ). Deuterium exchange studies on the partially structured apo-EhCaBP reveal that the C-terminal domain is better structured than the N-terminal domain. The protein can be reversibly folded and unfolded upon addition of Ca(2+) and EGTA, respectively. Titration shows a slow initial folding of the apo form with increasing Ca(2+) concentration, followed by a highly cooperative folding to its final state at a certain threshold of Ca(2+). Ca(2+) and the EGTA titration taken together show that site II in the N-terminal domain has the highest affinity for Ca(2+) contrary to earlier studies. Further, this study has thrown light on the relative Ca(2+) binding affinity and specificity of each site in the intact protein. A structural model for the partially collapsed form of apo-EhCaBP and its equilibrium folding to its completely folded holo state has been suggested. Large conformational changes seen in transforming from the apo to holo form of EhCaBP suggest that this protein should be functioning as a sensor protein and might have a significant role in host-parasite recognition.
Mukherjee
Sulakshana
S
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India.
Kuchroo
Kavita
K
Chary
Kandala V R
KV
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D002942:Circular Dichroism; D003903:Deuterium; D004748:Entamoeba histolytica / Q000378:metabolism*; D006859:Hydrogen; D008027:Light; D008969:Molecular Sequence Data; D017510:Protein Folding; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014867:Water / Q000378:metabolism
D001059:Apoproteins; D002135:Calcium-Binding Proteins; D015800:Protozoan Proteins; D014867:Water; D006859:Hydrogen; D003903:Deuterium; D002118:Calcium