Calcium regulates folding and disulfide-bond formation in α-lactalbumin

Rao, K.R.S. Sambasiva; Brew, Keith

doi:10.1016/0006-291x(89)91133-9

Cited by 62 publications

(45 citation statements)

References 15 publications

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“…Calcium accelerates the rate of refolding in denatured bLA (disulfide bonds intact) by more than 2 orders of magnitude (17,42); the dependence of folding kinetics on the Ca 2ϩ concentration is thought to result from calcium binding to high energy intermediates in the folding process which have partially formed Ca 2ϩ -binding sites (17). The requirement for Ca 2ϩ in the formation of native protein with correct disulfide bond arrangements from the reduced denatured protein (14,15) suggests that it also binds selectively to similar rate-limiting intermediates in the oxidative folding process. The MG and the high energy intermediates in folding are arrays of conformations as opposed to discrete species and Ca 2ϩ binding appears to be a common property of a range of partially folded states of LA in which affinity for the ion increases as their Ca 2ϩ -binding sites become more native.…”

Section: Resultsmentioning

confidence: 99%

“…The K d app for Ca 2ϩ binding to apo-LA under physiological conditions is of the order of 10 Ϫ7 M (11)(12)(13). The Ca 2ϩ ion in LA has a structural role, being required for folding and native disulfide bond formation in the reduced denatured protein (14,15). At ambient temperature and low ionic strength, apo-LA undergoes a transconformation to a molten globule (MG) state, which lacks a fixed tertiary structure but retains much of the native secondary structure.…”

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confidence: 99%

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Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Chrysina¹,

Brew²,

Acharya³

2000

Journal of Biological Chemistry

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High affinity binding of Ca2؉ to ␣-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca 2؉ as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca 2؉ . To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-Å resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of ␣-helical and ␤- ␣-Lactalbumin (LA)1 is the regulatory protein of lactose synthase (1-3), modulating the affinity of the catalytic component, UDP-galactose ␤-N-acetylglucosaminide ␤1,4-galactosyltransferase I, for acceptor substrates through a reversible proteinprotein interaction (4). LA is also secreted into milk and aggregates of a partially folded form of the human protein have been found to have selective apoptotic effects on tumor cells (5). LA is homologous with the c-type lysozymes (LZs) (6) and provides an example of extreme functional divergence in homologous proteins with closely similar structures (7-9). LA binds calcium strongly and specifically but the LZs include subgroups that bind Ca 2ϩ at a site corresponding to that in LA and others ("conventional" LZs) that lack a Ca 2ϩ -binding site (10). The K d app for Ca 2ϩ binding to apo-LA under physiological conditions is of the order of 10 Ϫ7 M (11-13). The Ca 2ϩ ion in LA has a structural role, being required for folding and native disulfide bond formation in the reduced denatured protein (14, 15). At ambient temperature and low ionic strength, apo-LA undergoes a transconformation to a molten globule (MG) state, which lacks a fixed tertiary structure but retains much of the native secondary structure. The MG is also stabilized by mildly disruptive treatments such as low pH or intermediate concentrations of denaturants (16,17); it is also identical to a kinetic MG that is highly populated during the refolding of chemically denatured LA (18 -20).Crystal structures of holo-LA from various species in different crystal forms and metal complexes (7,8,(21)(22)(23)(24)(25)(26) are similar to those of the homologous LZs, with two lobes of structure separated by a cleft (Fig. 1A). The larger ␣-helical lobe is formed by the amino-and carboxyl-terminal sections of the polypeptide chain (residues 1-34 and 86 -123) while the smaller lobe, which encompasses a small three-stranded antiparallel ␤-sheet, a small 3 10 helix and some irregular structure, is formed by the central section of the polypeptide chain (residues 35-85). We refer to the lobes as "subdomains" since they lack properties normally expected of true domains such as sequence contiguity and distinct functional properties and origins. The high affinity Ca 2ϩ -binding site is l...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Chrysina¹,

Brew²,

Acharya³

2000

Journal of Biological Chemistry

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240

217

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“…1). a-LA isolated from milk contains an equimolar amount of bound calcium, which stabilizes the native structure, but other divalent metal ions including Mn2+, Zn2+, and Cu2+ can also bind to the protein (Kronman et al, 198 1;Rao & Brew, 1989). The structure of a-LA consists of two domains, an a-helical and a P-sheet domain (Acharya et al, 1989, 1991Pike et al, 1996), and a calcium binding loop in which two carbonyl groups corresponding to K79 and D84, and three carboxyl groups corresponding to D82, D87, and D88, act as ligands.…”

Section: Introductionmentioning

confidence: 99%

Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr

Kim¹,

Baum²

1998

Protein Science

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Abstracta-Lactalbumin (a-LA) undergoes a pH-dependent unfolding from the native state to a partially unfolded state (the molten globule state). To understand the role of electrostatic interactions in protein denaturation, NMR and CD pH titration experiments are performed on guinea pig a-LA. Variation of pH over the range of 7.0 to 2.0 simultaneously leads to the acid denaturation of the protein and the titration of individual ionizable groups. The pH titrations are interpreted in the context of these coupled events, and indicate that acid denaturation in a-LA is a cooperative event that is triggered by the protonation of two ionizable residues. Our NMR results suggest that the critical electrostatic interactions that contribute to the denaturation of a-LA are concentrated in the calcium binding region of the protein.Keywords: chemical exchange; ionizable groups; NMR; pH titration; pK, values a-Lactalbumin (a-LA) offers an opportunity to study protein folding in the context of electrostatic interactions and metal binding (Hall & Campbell, 1986;McKenzie &White, 1991; Brew & Grobler, 1992;Sugai & Ikeguchi, 1994). Guinea pig a-LA contains 11 lysines, 4 histidines, 6 glutamic acids, and 16 aspartic acids that result in an isoelectric point of pH 4.65 (Fig. 1). a-LA isolated from milk contains an equimolar amount of bound calcium, which stabilizes the native structure, but other divalent metal ions including Mn2+, Zn2+, and Cu2+ can also bind to the protein (Kronman et al., 198 1;Rao & Brew, 1989). The structure of a-LA consists of two domains, an a-helical and a P-sheet domain (Acharya et al., 1989, 1991Pike et al., 1996), and a calcium binding loop in which two carbonyl groups corresponding to K79 and D84, and three carboxyl groups corresponding to D82, D87, and D88, act as ligands.a-LA undergoes a pH-dependent unfolding from the native state (N-state) to the acid denatured (A-state) or molten globule state (Kuwajima et al., Sommers & Kronman, 1980). The low pH form of a-LA is an ideal model system for studies of protein folding because the "molten globule" state that it adopts at low pH has been postulated to be analogous to an early intermediate on the protein folding pathway (Kuwajima et al., 1985;Ptitsyn et al., 1990). The low pH form of a-LA has been extensively studied by CD, NMR, and other spectroscopic and physicochemical methods (Dobson, 1994;Ptitsyn, 1995;Kuwajima, 1996)

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“…Ca 2ϩ ions are also implicated in the folding and disulfide bond formation of several proteins (49 -51). In the absence of Ca 2ϩ ions, negligible amounts of ␣-lactalbumin are refolded, whereas substantial renaturation is attained in the presence of Ca 2ϩ ions (49). Moreover, folding intermediates of ␣-lactalbumin contain only two native disulfide bonds in the presence of Ca 2ϩ ions and subsequently form the native structure when Ca 2ϩ ions bind to the ␤-sheet calcium-binding domain (50).…”

Section: Discussionmentioning

confidence: 99%

Dimerization of Matrix Metalloproteinase-2 (MMP-2)

Koo

Kim

Han

et al. 2012

Journal of Biological Chemistry

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Background: Matrix metalloproteinase-2 (MMP-2) activity is regulated by several mechanisms. Results: We observed that Ca 2ϩ ion is essential for MMP-2 homodimerization, which in turn results in the proteolysis of small peptide substrates and enhances thrombin-mediated activation of pro-MMP-2. Conclusion: Pro-MMP-2 activation is modulated by MMP-2 homodimerization. Significance: This study elucidates a novel mechanism to regulate MMP-2 activity.

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Calcium regulates folding and disulfide-bond formation in α-lactalbumin

Cited by 62 publications

References 15 publications

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Electrostatic interactions in the acid denaturation of α‐lactalbumin determined by nmr

Dimerization of Matrix Metalloproteinase-2 (MMP-2)

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