{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T05:05:12Z","timestamp":1768453512117,"version":"3.49.0"},"reference-count":33,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Biophys"],"published-print":{"date-parts":[[2012,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>EF-hand proteins can be activated by the binding of various heavy metals other than calcium, and such complexes can disturb the calcium-signaling pathway and cause toxicity and disease causing state. So far, no comprehensive study has been done to understand different heavy metals binding to calcium signaling proteins.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>In this work, the flexibility of the EF-hand motifs are examined by crystallographic and thermodynamic studies of binding of Pb<jats:sup>2+<\/jats:sup>, Ba<jats:sup>2+<\/jats:sup> and Sr<jats:sup>2+<\/jats:sup> to Calcium Binding Protein-1 from <jats:italic>Entamoeba histolytica<\/jats:italic> (EhCaBP1). The structures of the EhCaBP1- heavy metal complexes are found to be overall similar, nevertheless specific differences in metal coordination, and small differences in the coordination distances between the metal and the ligands in the metal binding loop. The largest such distances occur for the Ba<jats:sup>2+<\/jats:sup>- EhCaBP1 complex, where two bariums are bound with partial occupancy at the EF2 motif. Thermodynamic studies confirm that EhCaBP1 has five binding sites for Ba<jats:sup>2+<\/jats:sup> compared to four binding sites for the other metals. These structures and thermodynamic studies reveal that the EF-hand motifs can accommodate several heavy atoms with similar binding affinities. The binding of Ca<jats:sup>2+<\/jats:sup> to the 1<jats:sup>st<\/jats:sup>, 2<jats:sup>nd<\/jats:sup> and 4<jats:sup>th<\/jats:sup> sites and the binding of Ba<jats:sup>2+<\/jats:sup> to the 1<jats:sup>st<\/jats:sup>, 2<jats:sup>nd<\/jats:sup>, 4<jats:sup>th<\/jats:sup> and 5<jats:sup>th<\/jats:sup> sites are both enthalpically and entropically driven, whereas the binding of Sr<jats:sup>2+<\/jats:sup> to the 1<jats:sup>st<\/jats:sup>, 2<jats:sup>nd<\/jats:sup> and 4<jats:sup>th<\/jats:sup> sites are simply enthalpy driven, interestingly in agreement with ITC data, Sr<jats:sup>2+<\/jats:sup> do not coordinate with water in this structure. For all the metals, binding to the 3<jats:sup>rd<\/jats:sup> site is only entropy driven.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>Energetically, Ca<jats:sup>2+<\/jats:sup> is preferred in three sites, while in one site Ba<jats:sup>2+<\/jats:sup> has better binding energy. The Sr<jats:sup>2+<\/jats:sup>-coordination in the EF hand motifs is similar to that of the native Ca<jats:sup>2+<\/jats:sup> bound structure, except for the lack of water coordination. Sr<jats:sup>2+<\/jats:sup> coordination seems to be a pre-formed in nature since all seven coordinating atoms are from the protein itself, which also correlates with entropy contributions in Sr<jats:sup>2+<\/jats:sup> binding. These findings improve our understanding of metal association with calcium binding proteins and of metal induced conformational changes.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/2046-1682-5-15","type":"journal-article","created":{"date-parts":[[2012,8,20]],"date-time":"2012-08-20T10:15:48Z","timestamp":1345457748000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Flexibility of EF-hand motifs: structural and thermodynamic studies of Calcium Binding Protein-1 from Entamoeba histolytica with Pb2+, Ba2+, and Sr2+"],"prefix":"10.1186","volume":"5","author":[{"given":"Shivesh","family":"Kumar","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ejaz","family":"Ahmad","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sanjeev","family":"Kumar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rizwan Hasan","family":"Khan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samudrala","family":"Gourinath","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2012,8,20]]},"reference":[{"key":"34_CR1","doi-asserted-by":"publisher","first-page":"9915","DOI":"10.1073\/pnas.93.18.9915","volume":"93","author":"HT Cline","year":"1996","unstructured":"Cline HT, Witte S, Jones KW: Low lead levels stunt neuronal growth in a reversible manner. Proc Natl Acad Sci U S A. 1996, 93: 9915-9920. 10.1073\/pnas.93.18.9915.","journal-title":"Proc Natl Acad Sci U S A"},{"key":"34_CR2","doi-asserted-by":"publisher","first-page":"5","DOI":"10.1093\/brain\/awg014","volume":"126","author":"TI Lidsky","year":"2003","unstructured":"Lidsky TI, Schneider JS: Lead neurotoxicity in children: basic mechanisms and clinical correlates. Brain. 2003, 126: 5-19. 10.1093\/brain\/awg014.","journal-title":"Brain"},{"key":"34_CR3","doi-asserted-by":"publisher","first-page":"310","DOI":"10.1002\/1097-0274(200009)38:3<310::AID-AJIM10>3.0.CO;2-9","volume":"38","author":"P Apostoli","year":"2000","unstructured":"Apostoli P, Bellini A, Porru S, Bisanti L: The effect of lead on male fertility: a time to pregnancy (TTP) study. Am J Ind Med. 2000, 38: 310-315. 10.1002\/1097-0274(200009)38:3<310::AID-AJIM10>3.0.CO;2-9.","journal-title":"Am J Ind Med"},{"key":"34_CR4","doi-asserted-by":"publisher","first-page":"221","DOI":"10.1016\/j.reprotox.2005.01.007","volume":"20","author":"I Hernandez-Ochoa","year":"2005","unstructured":"Hernandez-Ochoa I, Garcia-Vargas G, Lopez-Carrill L, Rubio-Andrade M, Moran-Martinez J, Cebrian ME, Quintanilla-Vega B: Low lead environmental exposure alters semen quality and sperm chromatin condensation in northern Mexico. Reprod Toxicol. 2005, 20: 221-228. 10.1016\/j.reprotox.2005.01.007.","journal-title":"Reprod Toxicol"},{"key":"34_CR5","doi-asserted-by":"publisher","first-page":"191","DOI":"10.1111\/j.1749-6632.1987.tb48774.x","volume":"514","author":"MR Moore","year":"1987","unstructured":"Moore MR, Goldberg A, Yeung-Laiwah AA: Lead effects on the heme biosynthetic pathway. Relationship to toxicity. Ann N Y Acad Sci. 1987, 514: 191-203. 10.1111\/j.1749-6632.1987.tb48774.x.","journal-title":"Ann N Y Acad Sci"},{"key":"34_CR6","doi-asserted-by":"publisher","first-page":"507","DOI":"10.1023\/B:JOBB.0000008023.11211.13","volume":"35","author":"WD Atchison","year":"2003","unstructured":"Atchison WD: Effects of toxic environmental contaminants on voltage-gated calcium channel function: from past to present. J Bioenerg Biomembr. 2003, 35: 507-532.","journal-title":"J Bioenerg Biomembr"},{"key":"34_CR7","doi-asserted-by":"publisher","first-page":"490","DOI":"10.1007\/BF01977632","volume":"64","author":"SH Chao","year":"1990","unstructured":"Chao SH, Bu CH, Cheung WY: Activation of troponin C by Cd2+ and Pb2+. Arch Toxicol. 1990, 64: 490-496. 10.1007\/BF01977632.","journal-title":"Arch Toxicol"},{"key":"34_CR8","doi-asserted-by":"publisher","first-page":"72","DOI":"10.1016\/0167-4838(95)00223-5","volume":"1293","author":"JM Aramini","year":"1996","unstructured":"Aramini JM, Hiraoki T, Grace MR, Swaddle TW, Chiancone E, Vogel HJ: NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins. Biochim Biophys Acta. 1996, 1293: 72-82. 10.1016\/0167-4838(95)00223-5.","journal-title":"Biochim Biophys Acta"},{"key":"34_CR9","doi-asserted-by":"crossref","first-page":"6816","DOI":"10.1016\/S0021-9258(18)88853-6","volume":"260","author":"CS Fullmer","year":"1985","unstructured":"Fullmer CS, Edelstein S, Wasserman RH: Lead-binding properties of intestinal calcium-binding proteins. J Biol Chem. 1985, 260: 6816-6819.","journal-title":"J Biol Chem"},{"key":"34_CR10","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1007\/s002040050158","volume":"69","author":"SH Chao","year":"1995","unstructured":"Chao SH, Bu CH, Cheung WY: Stimulation of myosin light-chain kinase by Cd2+ and Pb2+. Arch Toxicol. 1995, 69: 197-203. 10.1007\/s002040050158.","journal-title":"Arch Toxicol"},{"key":"34_CR11","doi-asserted-by":"publisher","first-page":"61","DOI":"10.1007\/BF00277816","volume":"54","author":"E Habermann","year":"1983","unstructured":"Habermann E, Crowell K, Janicki P: Lead and other metals can substitute for Ca2+ in calmodulin. Arch Toxicol. 1983, 54: 61-70. 10.1007\/BF00277816.","journal-title":"Arch Toxicol"},{"key":"34_CR12","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1016\/0014-5793(79)81097-2","volume":"104","author":"S Forsen","year":"1979","unstructured":"Forsen S, Thulin E, Lilja H: 113Cd NMR in the study of calcium binding proteins: troponin C. FEBS Lett. 1979, 104: 123-126. 10.1016\/0014-5793(79)81097-2.","journal-title":"FEBS Lett"},{"key":"34_CR13","doi-asserted-by":"publisher","first-page":"479","DOI":"10.1007\/s007750100214","volume":"6","author":"E Pidcock","year":"2001","unstructured":"Pidcock E, Moore GR: Structural characteristics of protein binding sites for calcium and lanthanide ions. J Biol Inorg Chem. 2001, 6: 479-489. 10.1007\/s007750100214.","journal-title":"J Biol Inorg Chem"},{"key":"34_CR14","doi-asserted-by":"publisher","first-page":"6410","DOI":"10.1021\/bi00321a020","volume":"23","author":"CL Wang","year":"1984","unstructured":"Wang CL, Leavis PC, Gergely J: Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2\u2009+\u2009\u2212binding sites of calmodulin. Biochemistry. 1984, 23: 6410-6415. 10.1021\/bi00321a020.","journal-title":"Biochemistry"},{"key":"34_CR15","doi-asserted-by":"publisher","first-page":"2085","DOI":"10.1021\/ja0431307","volume":"127","author":"W Yang","year":"2005","unstructured":"Yang W, Wilkins AL, Ye Y, Liu ZR, Li SY, Urbauer JL, Hellinga HW, Kearney A, van der Merwe PA, Yang JJ: Design of a calcium-binding protein with desired structure in a cell adhesion molecule. J Am Chem Soc. 2005, 127: 2085-2093. 10.1021\/ja0431307.","journal-title":"J Am Chem Soc"},{"key":"34_CR16","doi-asserted-by":"crossref","first-page":"20817","DOI":"10.1016\/S0021-9258(19)47135-4","volume":"264","author":"MN Vyas","year":"1989","unstructured":"Vyas MN, Jacobson BL, Quiocho FA: The calcium-binding site in the galactose chemoreceptor protein. Crystallographic and metal-binding studies. J Biol Chem. 1989, 264: 20817-20821.","journal-title":"J Biol Chem"},{"key":"34_CR17","doi-asserted-by":"publisher","first-page":"653","DOI":"10.1107\/S1744309107034525","volume":"63","author":"P Kursula","year":"2007","unstructured":"Kursula P, Majava V: A structural insight into lead neurotoxicity and calmodulin activation by heavy metals. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007, 63: 653-656.","journal-title":"Acta Crystallogr Sect F Struct Biol Cryst Commun"},{"key":"34_CR18","doi-asserted-by":"publisher","first-page":"1782","DOI":"10.1107\/S0907444903016846","volume":"59","author":"MA Wilson","year":"2003","unstructured":"Wilson MA, Brunger AT: Domain flexibility in the 1.75 A resolution structure of Pb2+\u2212calmodulin. Acta Crystallogr D: Biol Crystallogr. 2003, 59: 1782-1792. 10.1107\/S0907444903016846.","journal-title":"Acta Crystallogr D: Biol Crystallogr"},{"key":"34_CR19","doi-asserted-by":"publisher","first-page":"990","DOI":"10.1002\/prot.21455","volume":"68","author":"S Kumar","year":"2007","unstructured":"Kumar S, Padhan N, Alam N, Gourinath S: Crystal structure of calcium binding protein-1 from Entamoeba histolytica: a novel arrangment of EF hand motifs. Proteins. 2007, 68: 990-998. 10.1002\/prot.21455.","journal-title":"Proteins"},{"key":"34_CR20","doi-asserted-by":"publisher","first-page":"2933","DOI":"10.1016\/j.bpj.2010.03.048","volume":"98","author":"S Kumar","year":"2010","unstructured":"Kumar S, Ahmad E, Mansuri MS, Kumar S, Jain R, Khan RH, Gourinath S: Crystal structure and trimer-monomer transition of N-terminal domain of EhCaBP1 from Entamoeba histolytica. Biophys J. 2010, 98: 2933-2942. 10.1016\/j.bpj.2010.03.048.","journal-title":"Biophys J"},{"key":"34_CR21","doi-asserted-by":"publisher","first-page":"10910","DOI":"10.1021\/bi9702546","volume":"36","author":"B Gopal","year":"1997","unstructured":"Gopal B, Swaminathan CP, Bhattacharya S, Bhattacharya A, Murthy MR, Surolia A: Thermodynamics of metal ion binding and denaturation of a calcium binding protein from Entamoeba histolytica. Biochemistry. 1997, 36: 10910-10916. 10.1021\/bi9702546.","journal-title":"Biochemistry"},{"key":"34_CR22","doi-asserted-by":"publisher","first-page":"2201","DOI":"10.1021\/bi992399d","volume":"39","author":"A Velazquez-Campoy","year":"2000","unstructured":"Velazquez-Campoy A, Todd MJ, Freire E: HIV-1 protease inhibitors: enthalpic versus entropic optimization of the binding affinity. Biochemistry. 2000, 39: 2201-2207. 10.1021\/bi992399d.","journal-title":"Biochemistry"},{"key":"34_CR23","doi-asserted-by":"publisher","first-page":"238","DOI":"10.1006\/jmbi.1997.1257","volume":"27","author":"NC Strynadka","year":"1997","unstructured":"Strynadka NC, Cherney M, Sielecki AR, Li MX, Smillie LB, James MN: Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 A resolution. J Mol Biol. 1997, 27: 238-255.","journal-title":"J Mol Biol"},{"key":"34_CR24","doi-asserted-by":"publisher","first-page":"e5269","DOI":"10.1371\/journal.pone.0005269","volume":"4","author":"R Jain","year":"2009","unstructured":"Jain R, Kumar S, Gourinath S, Bhattacharya S, Bhattacharya A: N- and C-terminal domains of the calcium binding protein EhCaBP1 of the parasite Entamoeba histolytica display distinct functions. PLoS One. 2009, 4: e5269-10.1371\/journal.pone.0005269.","journal-title":"PLoS One"},{"key":"34_CR25","volume-title":"The AUTOMAR Manual, v.1.4","author":"KS Bartels","year":"2003","unstructured":"Bartels KS, Klein C: The AUTOMAR Manual, v.1.4. 2003, Norderstedt, Germany: MAR Research GmbH"},{"key":"34_CR26","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1254\/jjp.70.351","volume":"70","author":"J Yamazaki","year":"1996","unstructured":"Yamazaki J, Urushidani T, Nagao T: Barium activates rat cerebellar nitric oxide synthase. Jpn J Pharmacol. 1996, 70: 351-354. 10.1254\/jjp.70.351.","journal-title":"Jpn J Pharmacol"},{"key":"34_CR27","doi-asserted-by":"publisher","first-page":"307","DOI":"10.1016\/S0076-6879(97)76066-X","volume":"276","author":"O Otwinowski","year":"1997","unstructured":"Otwinowski O, Minor W: Processing of X-ray diffraction data collected in oscillation mode. Meth Enzymol. 1997, 276: 307-326.","journal-title":"Meth Enzymol"},{"key":"34_CR28","doi-asserted-by":"publisher","first-page":"432","DOI":"10.1107\/S0907444903028956","volume":"60","author":"LC Storoni","year":"2004","unstructured":"Storoni LC, McCoy AJ, Read RJ: Likelihood-enhanced fast rotation functions. Acta Crystallogr D: Biol Crystallogr. 2004, 60: 432-438. 10.1107\/S0907444903028956.","journal-title":"Acta Crystallogr D: Biol Crystallogr"},{"key":"34_CR29","doi-asserted-by":"publisher","first-page":"2126","DOI":"10.1107\/S0907444904019158","volume":"60","author":"P Emsley","year":"2004","unstructured":"Emsley P, Cowtan K: Coot: model-building tools for molecular graphics. Acta Crystallogr D: Biol Crystallogr. 2004, 60: 2126-2132. 10.1107\/S0907444904019158.","journal-title":"Acta Crystallogr D: Biol Crystallogr"},{"key":"34_CR30","doi-asserted-by":"publisher","first-page":"905","DOI":"10.1107\/S0907444998003254","volume":"54","author":"AT Brunger","year":"1998","unstructured":"Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL: Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D: Biol Crystallogr. 1998, 54: 905-921. 10.1107\/S0907444998003254.","journal-title":"Acta Crystallogr D: Biol Crystallogr"},{"key":"34_CR31","doi-asserted-by":"publisher","first-page":"897","DOI":"10.1016\/S0969-2126(96)00097-4","volume":"4","author":"GJ Kleywegt","year":"1996","unstructured":"Kleywegt GJ, Brunger AT: Checking your imagination: applications of the free R value. Structure. 1996, 4: 897-904. 10.1016\/S0969-2126(96)00097-4.","journal-title":"Structure"},{"key":"34_CR32","doi-asserted-by":"publisher","first-page":"1437","DOI":"10.1107\/S0907444905023589","volume":"61","author":"PH Zwart","year":"2005","unstructured":"Zwart PH: Anomalous signal indicators in protein crystallography. Acta Crystallogr D: Biol Crystallogr. 2005, 61: 1437-1448. 10.1107\/S0907444905023589.","journal-title":"Acta Crystallogr D: Biol Crystallogr"},{"key":"34_CR33","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1016\/S0014-5793(98)01513-0","volume":"441","author":"B Gopal","year":"1998","unstructured":"Gopal B, Krishna Rao JV, Thomas CJ, Bhattacharya A, Bhattacharya S, Murthy MR, Surolia A: Induction of a spectroscopically defined transition by guanidinium hydrochloride on a recombinant calcium binding protein from Entamoeba histolytica. FEBS Lett. 1998, 441: 71-76. 10.1016\/S0014-5793(98)01513-0.","journal-title":"FEBS Lett"}],"container-title":["BMC Biophysics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/2046-1682-5-15.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,9,1]],"date-time":"2021-09-01T20:09:36Z","timestamp":1630526976000},"score":1,"resource":{"primary":{"URL":"https:\/\/bmcbiophys.biomedcentral.com\/articles\/10.1186\/2046-1682-5-15"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2012,8,20]]},"references-count":33,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2012,12]]}},"alternative-id":["34"],"URL":"https:\/\/doi.org\/10.1186\/2046-1682-5-15","relation":{},"ISSN":["2046-1682"],"issn-type":[{"value":"2046-1682","type":"electronic"}],"subject":[],"published":{"date-parts":[[2012,8,20]]},"assertion":[{"value":"30 April 2012","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"9 August 2012","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"20 August 2012","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}],"article-number":"15"}}