The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 A resolution.

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.     

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

The backbone resonance assignments have been completed for the apo (1H and 15N) and calcium-loaded (1H, 15N, 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 3JHNH alpha coupling constants, permitted the determination and quantification of the Ca(2+)-induced secondary structural change in the N-domain of TnC. For both structures, 5 helices and 2 short beta-strands were found, as was observed in the apo N-domain of the crystal structure of whole TnC (Herzberg O, James MNG, 1988, J Mol 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 Ca(2+)-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 alpha-helical content associated with the transition from apo to the 2Ca2+ state of the N-domain of TnC. Therefore, the Ca(2+)-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.     

A structural basis for S100 protein specificity derived from comparative analysis of apo and Ca(2+)-calcyclin

Calcyclin is a homodimeric protein belonging to the S100 subfamily of EF-hand Ca(2+)-binding proteins, which function in Ca(2+) signal transduction processes. A refined high-resolution solution structure of Ca(2+)-bound rabbit calcyclin has been determined by heteronuclear solution NMR. In order to understand the Ca(2+)-induced structural changes in S100 proteins, in-depth comparative structural analyses were used to compare the apo and Ca(2+)-bound states of calcyclin, the closely related S100B, and the prototypical Ca(2+)-sensor protein calmodulin. Upon Ca(2+) binding, the position and orientation of helix III in the second EF-hand is altered, whereas the rest of the protein, including the dimer interface, remains virtually unchanged. This Ca(2+)-induced structural change is much less drastic than the "opening" of the globular EF-hand domains that occurs in classical Ca(2+) sensors, such as calmodulin. Using homology models of calcyclin based on S100B, a binding site in calcyclin has been proposed for the N-terminal domain of annexin XI and the C-terminal domain of the neuronal calcyclin-binding protein. The structural basis for the specificity of S100 proteins is discussed in terms of the variation in sequence of critical contact residues in the common S100 target-binding site.     

Solution structures of the C-terminal domain of cardiac troponin C free and bound to the N-terminal domain of cardiac troponin I.

The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex.     

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations

To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.     

Binding of cardiac troponin-I147-163 induces a structural opening in human cardiac troponin-C.

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.     

Refined crystal structure of troponin C from turkey skeletal muscle at 2.0 A resolution

The crystal structure of troponin C from turkey skeletal muscle has been refined at 2.0 A resolution (1 A = 0.1 nm). The resulting crystallographic R factor (R = sigma[[Fo[-[Fc[[/sigma[Fo[, where [Fo[ and [Fc[ are the observed and calculated structure factor amplitudes) is 0.155 for the 8054 reflections with intensities I greater than or equal to 2 sigma(I) within the 10 A to 2.0 A resolution range. With 66% of the residues in helical conformation, troponin C provides a good sample for helix analysis. The mean alpha-helix dihedral angles (phi, psi = -62 degrees, -42 degrees) agree with values observed for helical regions in other proteins. The helices are all curved and/or kinked. In particular, the 31 amino acid long inter-domain helix is smoothly curved, with a rather large radius of curvature of 137 A. Helix packing is different in the Ca2+-free domain (N-terminal) and the Ca2+-bound domain (C-terminal). The inter-helix angles for the two helix-loop-helix motifs in the regulatory domain are 133 degrees and 151 degrees, whereas the value for the two motifs in the C-terminal domain is 110 degrees, as observed in the EF-hands of parvalbumin. These differences affect the packing of the respective hydrophobic cores of each domain, in particular the disposition of aromatic rings. Pairwise arrangement of Ca2+-binding loops is common to both states, but the conformation is markedly different. Conversion of one to the other can be achieved by small cumulative changes of main-chain dihedral angles. The integrity of loop structure is maintained by numerous electrostatic interactions. Both salt bridges and carboxyl-carboxylate interactions are observed in TnC. There are more intramolecular (9) than intermolecular (1) salt bridges. Carboxyl-carboxylate interactions occur because the pH of the crystals is 5.0 and there is a multitude of aspartate and glutamate residues. One is intramolecular and four are intermolecular. Polar side-chain interactions occur more commonly with main-chain carbonyls and amides than with other polar side-chains. These interactions are mostly short range, and are similar to those observed in other proteins with one exception: negatively charged side-chains interact more frequently with main-chain carbonyl oxygen atoms. However, out of 19 such interactions, 10 involve oxygen atoms of the Ca2+ ligands. These unfavorable interactions are compensated by the favorable interactions with the Ca2+ ions and with main-chain amides. They are a trivial consequence of the tight fold of the Ca2+-binding loops.     

Conformation of the regulatory domain of cardiac muscle troponin C in its complex with cardiac troponin I.

Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca(2+)-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca(2+). However, in the presence of bound cardiac TnI (cTnI), activator Ca(2+) induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC.cTnI complex has properties favorable for the Ca(2+)-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca(2+)-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship.     

Kinetics of conformational transitions in cardiac troponin induced by Ca2+ dissociation determined by Förster resonance energy transfer

Upon Ca2+ activation of cardiac muscle, several structural changes occur in the troponin subunits. These changes include the opening of the cardiac troponin C (cTnC) N-domain, the change of secondary structure of the inhibitory region of cardiac troponin I (cTnI), and the change in the separation between these two proteins in the cTnC-cTnI interface. We have used F?rster resonance energy transfer in Ca2+ titration and stopped-flow experiments to delineate these transitions using a reconstituted cardiac troponin. Energy transfer results were quantified to yield time-dependent profiles of changes in intersite distances during Ca2+ dissociation. The closing of the cTnC N-domain induced by release of regulatory Ca2+ from cTnC occurs in one step (t1/2 approximately 5 ms), and this transition is not affected by Ca2+ release from the C-domain. The other two transitions triggered by Ca2+ dissociation are biphasic with the fast phase (t1/2 approximately 5 ms) correlated with Ca2+ release from the cTnC N-domain. These transitions are slower than the release of bound regulatory Ca2+ (t1/2 3.6 ms) and are coupled to one another in a cooperative manner in restoring their conformations in the deactivated state. The kinetic results define the magnitudes of structural changes relevant in Ca2+ switching between activation and deactivation of cardiac muscle contraction.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca²⁺ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.     

NMR structures of two variants of bovine pancreatic trypsin inhibitor (BPTI) reveal unexpected influence of mutations on protein structure and stability

Here we determined NMR solution structures of two mutants of bovine pancreatic trypsin inhibitor (BPTI) to reveal structural reasons of their decreased thermodynamic stability. A point mutation, A16V, in the solvent-exposed loop destabilizes the protein by 20 degrees C, in contrast to marginal destabilization observed for G, S, R, L or W mutants. In the second mutant introduction of eight alanine residues at proteinase-contacting sites (residues 11, 13, 17, 18, 19, 34, 37 and 39) provides a protein that denatures at a temperature about 30 degrees C higher than expected from additive behavior of individual mutations. In order to efficiently determine structures of these variants, we applied a procedure that allows us to share data between regions unaffected by mutation(s). NOAH/DYANA and CNS programs were used for a rapid assignment of NOESY cross-peaks, structure calculations and refinement. The solution structure of the A16V mutant reveals no conformational change within the molecule, but shows close contacts between V16, I18 and G36/G37. Thus, the observed 4.3kcal/mol decrease of stability results from a strained local conformation of these residues caused by introduction of a beta-branched Val side-chain. Contrary to the A16V mutation, introduction of eight alanine residues produces significant conformational changes, manifested in over a 9A shift of the Y35 side-chain. This structural rearrangement provides about 6kcal/mol non-additive stabilization energy, compared to the mutant in which G37 and R39 are not mutated to alanine residues.     

Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 A resolution.

The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.     

Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms

The stimulation of 2-oxoglutarate and NAD(+)-isocitrate dehydrogenase by Ca2+ in mitochondria from normal tissues has been proposed to mediate partially the activation of oxidative energy metabolism elicited by physiological elevations in cytosolic Ca2+. This mode of regulation may also occur in tumor cells in which several aspects of mitochondrial metabolism are known to be altered. This study provides a comparison of the stimulation by submicromolar concentrations of Ca2+ on the rates of ATP-generating (state 3) respiration under physiologically realistic conditions by mitochondria isolated from normal rat liver and from highly malignant rat AS-30D ascites hepatoma cells. The K0.5 for activation of glutamate-dependent state 3 respiration by Ca2+ in the presence of ATP at 37 degrees C was determined to be 0.70 +/- 0.05 (S.E.) microM for hepatoma mitochondria and 0.90 +/- 0.03 microM for rat liver mitochondria. This activation was also reflected by a Ca2(+)-induced shift in the oxidation-reduction state of hepatoma mitochondrial pyridine nucleotides to a more reduced level and Ca2+ stimulation of 14CO2 production from [1-14C]glutamate. Whereas the Ca2+ sensitivity of state 3 respiration by hepatoma mitochondria can be explained by the activation of 2-oxoglutarate and possibly NAD(+)-isocitrate dehydrogenases, the Ca2+ sensitivity of liver mitochondrial respiration appears to be predominantly mediated by activation of electron flow through ubiquinone and Complex III of the electron transport chain, as indicated by the specificity of the effects of Ca2+ on respiration with different oxidizable substrates. Although rat liver and hepatoma mitochondria employ different modes of Ca2(+)-activated ATP generation, these results support the hypothesis that changes in cytosolic Ca2+ play a significant role in the potentiation of energy production in tumor, as well as normal tissue.     

Designing calcium-sensitizing mutations in the regulatory domain of cardiac troponin C

Cardiac troponin C belongs to the EF-hand superfamily of calcium-binding proteins and plays an essential role in the regulation of muscle contraction and relaxation. To follow calcium binding and exchange with the regulatory N-terminal domain (N-domain) of human cardiac troponin C, we substituted Phe at position 27 with Trp, making a fluorescent cardiac troponin C(F27W). Trp(27) accurately reported the kinetics of calcium association and dissociation of the N-domain of cardiac troponin C(F27W). To sensitize the N-domain of cardiac troponin C(F27W) to calcium, we individually substituted the hydrophobic residues Phe(20), Val(44), Met(45), Leu(48), and Met(81) with polar Gln. These mutations were designed to increase the calcium affinity of the N-domain of cardiac troponin C by facilitating the movement of helices B and C (BC unit) away from helices N, A, and D (NAD unit). As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C(F27W) approximately 2.1-15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6-8.7-fold faster) rather than to slower calcium dissociation rates (1.2-2.9-fold slower). The regulatory N-domains of cardiac troponin C(F27W) and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2-2.7 mm). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.     

Ca2+-induced conformational transition in the inhibitory and regulatory regions of cardiac troponin I

Cardiac muscle activation is initiated by the binding of Ca(2+) to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca(2+) binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using F?rster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg(2+)-saturated) and Ca(2+)-activated (Ca(2+)-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca(2+). Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca(2+)-induced increases of >8 A. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca(2+) activation causes residues within cTnI-I to switch from a beta-turn/coil to an extended quasi-alpha-helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha-helical in both Mg(2+)- and Ca(2+)-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg(2+)- and Ca(2+)-saturated states.     

Peptides related to the active fragment of "proline rich polypeptide", an immunoregulatory protein of the ovine colostrum. Spectroscopic and computer modeling studies

The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some of its structural analogues was investigated by NMR-spectroscopy, spectrofluorimetry and computer simulation technic. It was found that the preferred conformation is characterized by cis'-conformation of Pro3 and the gamma-turn on the Leu4-residue: for Val2 and Phe5 a beta-structure seems to be privileged. In such a conformation Val2 and Leu4 residues occupy exactly the same positions in space as residues i and i + 3 in an alpha-helix. It suggests that the PRP-hexapeptide can interact with receptor protein inducing or stabilizing its helical conformation by "knobs into holes" packing.     

The EF-hand domain: A globally cooperative structural unit

EF-hand Ca(2+)-binding proteins participate in both modulation of Ca(2+) signals and direct transduction of the ionic signal into downstream biochemical events. The range of biochemical functions of these proteins is correlated with differences in the way in which they respond to the binding of Ca(2+). The EF-hand domains of calbindin D(9k) and calmodulin are homologous, yet they respond to the binding of calcium ions in a drastically different manner. A series of comparative analyses of their structures enabled the development of hypotheses about which residues in these proteins control the calcium-induced changes in conformation. To test our understanding of the relationship between protein sequence and structure, we specifically designed the F36G mutation of the EF-hand protein calbindin D(9k) to alter the packing of helices I and II in the apoprotein. The three-dimensional structure of apo F36G was determined in solution by nuclear magnetic resonance spectroscopy and showed that the design was successful. Surprisingly, significant structural perturbations also were found to extend far from the site of mutation. The observation of such long-range effects provides clear evidence that four-helix EF-hand domains should be treated as a single globally cooperative unit. A hypothetical mechanism for how the long-range effects are transmitted is described. Our results support the concept of energetic and structural coupling of the key residues that are crucial for a protein's fold and function.     

Crystal structure of human CD38 extracellular domain

Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 A resolution. The enzyme's overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38's multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.     

The enzymatic activity of proteinase K is controlled by calcium

The fungal proteinase K (EC 3.4.21.14) is a very potent unusually stable member of the subtilisin family. Its X-ray structure determined at 0.15-nm resolution shows two bound Ca2+ ions. Ca1 is in near-ideal pentagonal bipyramidal configuration with Asp200 carboxylate and Pro175 peptide C = O in an apical, and Val177 peptide C = O and four water molecules in an equatorial position, whereas Ca2 displays incomplete octahedral coordination with the carboxylate of Asp260, the peptide C = O of Val16 and the two water molecules. Scatchard analysis of the titration of Ca2+-free proteinase K with Ca2+ yields a single dissociation constant (7.6 +/- 2.5) x 10(-8) M associated with the tightly bound Ca1 whereas Ca2 is so weakly bound that it cannot be titrated. If proteinase K is depleted of Ca2+ by treatment with EDTA, followed by gel filtration, its enzymatic activity drops within 6 h to 20% of its original value, without autolysis. Addition of excess Ca2+ immediately raises the residual activity to 28%, but full activity is not achieved. Removal of Ca2+ triggers a conformational change of the substrate recognition site because there is a direct connection, via secondary structure hydrogen bonds, between the Ca1 binding site and the substrate-recognition site. This is indicated further by circular dichroism and fluorescence-spectroscopic data, and by reversed-phase FPLC, carried out in the presence and absence of Ca2+, but the overall structure of the enzyme is not affected. Depletion of Ca2+ also influences binding of longer peptide inhibitors of the chloromethane type, it increases the rate of autolysis after about 48 h, it reduces the thermal stability (measured by activity tests from 65 degrees C to 46 degrees C), and it enhances the deactivation by 8 M urea which inactivates to only 65%, whereas sodium dodecyl sulfate totally inactivates at a concentration of 12.5%.