Calcium-Binding Sites in Proteins: A Structural Perspective

McPhalen, Catherine A.; Strynadka, N.C.J.; James, Michael N.G.

doi:10.1016/s0065-3233(08)60535-5

Cited by 262 publications

(266 citation statements)

References 133 publications

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“…The kink at residue 41, sult in an increase of symmetry between the 2 binding sites upon which was well characterized in the crystal structure of chicken addition of calcium. This is in agreement with the model proposed by Herzberg et al (1986) and many helix-loop-helix protein structures, which were solved in the Ca2+-saturated state either by X-ray crystallography (see reviews by Strynadka & James, 1989;McPhalen et al, 1991) or NMR (Akke et al, 1992; ring in the 2 Ca2+-binding loops are not presented here in detail and must await the determination of the tertiary structure of NTnC .2Ca, the straightening of helix-B is the major Ca2+-induced structural change we can assess at this time. This change is localized at E41, a position that corresponds to the third most conserved residue in calcium-binding sites of proteins having the helix-loop-helix motif (Marsden et al, 1990).…”

Section: Discussionsupporting

confidence: 91%

Quantification of the calcium‐induced secondary structural changes in the regulatory domain of troponin‐C

et al. 1994

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The backbone resonance assignments have been completed for the apo ('H and "N) and calcium-loaded ('H, IsN, and 13C) regulatory N-domain of chicken skeletal troponin-C (1-90), using multidimensional homonuclear and heteronuclear NMR spectroscopy. The chemical-shift information, along with detailed NOE analysis and 3JHNHa coupling constants, permitted the determination and quantification of the Ca2+-induced secondary structural change in the N-domain of TnC. For both structures, 5 helices and 2 short 0-strands were found, as was observed in the apo N-domain of the crystal structure of whole TnC (Herzberg 0, James MNG, 1988, JMol Biol 203:761-779). The NMR solution structure of the apo form is indistinguishable from the crystal structure, whereas some structural differences are evident when comparing the 2Ca2+ state solution structure with the apo one. The major conformational change observed is the straightening of helix-B upon Ca2+ binding. The possible importance and role of this conformational change is explored. Previous CD studies on the regulatory domain of TnC showed a significant Ca2+-induced increase in negative ellipticity, suggesting a significant increase in helical content upon Ca2+ binding. The present study shows that there is virtually no change in a-helical content associated with the transition from apo to the 2Ca2+ state of the N-domain of TnC. Therefore, the Ca2+-induced increase in ellipticity observed by CD does not relate to a change in helical content, but more likely to changes in spatial orientation of helices.Keywords: calcium; CD; NMR; regulatory domain of troponin-C; secondary structural change Troponin-C has a key role in muscle contraction of vertebrate striated muscle (skeletal and cardiac). The binding of Ca2+ to TnC induces a conformational change that affects the interaction between TnC, troponin-I (TnI), and troponin-T (TnT). This interaction blocks the inhibitory action of TnI, allowing formation of the Mg2+-activated ATPase actomyosin complex, and ultimately leads to muscle contraction. The roles and interactions of proteins in the regulatory system of striated muscle have been studied extensively (Leavis & Gergely, 1984; Ohtsuki et al.,

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Section: Discussionsupporting

confidence: 91%

Quantification of the calcium‐induced secondary structural changes in the regulatory domain of troponin‐C

et al. 1994

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“…The Ca2+-oxygen distances are 2,4-2.5 A, i.e. only slightly exceed mean values of Ca2+-carbonyl interactions in protein structures (2.36 A [33]). The B value of this Ca 2+ refines to 15 A 2, comparable to that of the coordinating carbonyl oxygens.…”

Section: Resultsmentioning

confidence: 70%

“…Due to a slight kink at this Pro residue, the preceding fragments deviate slightly from one another, with both fragments exhibiting extended conformations. 33 Structural analysis of the MMP2-CTD reveals that it is topologically quite similar to that of porcine MMP1. The only significant conformational differences occur at the exit side of the disc, although both entry and exit sides display different charge patterns.…”

Section: Resultsmentioning

confidence: 96%

The C‐terminal (haemopexin‐like) domain structure of human gelatinase A (MMP2): structural implications for its function

Gohlke

Gomis-Rüth

Crabbe³

et al. 1996

FEBS Letters

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In common with most other matrix metalloproteinases, gelatinase A has a non-catalytic C-terminal domain that displays sequence homology to haemopexin. Crystals of this domain were used by molecular replacement to solve its molecular structure at 2.6 A resolution, which was refined to an R value of 17.9%. This structure has a discqike shape, with the chain folded into a/3-propeller structure that has pseudo four-fold symmetry. Although the topology and the side-chain arrangement are very similar to the equivalent domain of fibroblast collagenase, significant differences in surface charge and contouring are observable on 1 side of the gelatinase A disc. This difference might be a factor in allowing the gelatinase A C-terminal domain to bind to natural inhibitor TIMP-2.

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“…If Ca 2+ indeed substitutes functionally for the side chain of Arg69, as has been demonstrated in the conservative R69 mutants (R69C-AA and R69C-TAA) (28), the presence of Ca 2+ should restore some of the stereoselectivity lost in R69D. To test this hypothesis, we determined the stereoselectivity of R69D in the presence and absence of Ca 2+ by 31 P NMR. The data, along with those from previous studies (28), are also summarized in Table 3.…”

Section: Construction Of a Mutant Showing Activation By Camentioning

confidence: 89%

“…The maximal velocities of R69D complexes with Mg 2+ , Ca 2+ , and Sr 2+ at saturating substrate concentration (2 mM) were 10.2 ( 0.2, 12.0 ( 0.3, and 2.7 ( 0.3 U/mg, which give a 35-, 41-, and 9-fold activation, respectively. The results described above can be considered successful for a de novo metal binding site (31). For comparison, mPI-PLC was activated by Ca 2+ at lower (34).…”

Section: Construction Of a Mutant Showing Activation By Camentioning

confidence: 89%

Engineering a Catalytic Metal Binding Site into a Calcium-Independent Phosphatidylinositol-Specific Phospholipase C Leads to Enhanced Stereoselectivity

et al. 2003

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Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69. In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties. The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca 2+ , and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca 2+ , monitored by 15 N-1 H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca 2+ to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca 2+ activation, the thio effect of the S Pisomer of the phosphorothioate analogue (k O /k Sp ) 4.4 × 10 5 ) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca 2+ ). The R P -thio effect (k O /k Rp ) 9.4) is smaller than that of the WT enzyme by a factor of 5. Consequently, R69D-Ca 2+ displays higher stereoselectivity (k Rp /k Sp ) 47 000) than WT (k Rp / k Sp ) 7600). (4) Results from additional mutagenesis analyses suggest that the Ca 2+ binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.Engineering of metal binding sites in proteins (reviewed in refs 1-3) has been used to address a variety of problems such as protein-protein interactions and topology of transmembrane domains (4, 5), regulation of enzyme activity and specificity (6, 7), and modification of enzyme redox chemistry (8). Surprisingly, the interchange between positively charged amino acid side chains and metal ions was successfully achieved only in the metal to amino acid direction (9

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Calcium-Binding Sites in Proteins: A Structural Perspective

Cited by 262 publications

References 133 publications

Quantification of the calcium‐induced secondary structural changes in the regulatory domain of troponin‐C

Quantification of the calcium‐induced secondary structural changes in the regulatory domain of troponin‐C

The C‐terminal (haemopexin‐like) domain structure of human gelatinase A (MMP2): structural implications for its function

Engineering a Catalytic Metal Binding Site into a Calcium-Independent Phosphatidylinositol-Specific Phospholipase C Leads to Enhanced Stereoselectivity

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