Impact of calcium binding and thionylation of S100A1 protein on its nuclear magnetic resonance-derived structure and backbone dynamics.

S100 proteins play a crucial role in multiple important biological processes in vertebrate organisms acting predominantly as calcium signal transmitters. S100A1 is a typical representative of this family of proteins. After four Ca(2+) ions bind, it undergoes a dramatic conformational change, resulting in exposure, in each of its two identical subunits, a large hydrophobic cleft that binds to target proteins. It has been shown that abnormal expression of S100A1 is strongly correlated with a number of severe human diseases: cardiomyopathy and neurodegenerative disorders. A few years ago, we found that thionylation of Cys 85, the unique cysteine in two identical S100A1 subunits, leads to a drastic increase of the affinity of the protein for calcium. We postulated that the protein activated by thionylation becomes a more efficient calcium signal transmitter. Therefore, we decided to undertake, using nuclear magnetic resonance methods, a comparative study of the structure and dynamics of native and thionylated human S100A1 in its apo and holo states. In this paper, we present the results obtained for both forms of this protein in its holo state and compare them with the previously published structure of native apo-S100. The main conclusion that we draw from these results is that the increased calcium binding affinity of S100A1 upon thionylation arises, most probably, from rearrangement of the hydrophobic core in its apo form.

Solution NMR structure and dynamics of human apo-S100A1 protein.

S100A1 belongs to the EF-hand superfamily of calcium binding proteins. It is a representative of the S100 protein family based on amino acid sequence, three-dimensional structure, and biological function as a calcium signal transmitter. It is a homodimer of noncovalently bound subunits. S100A1, like most of other members of the S100 protein family, is a multifunctional, regulatory protein involved in a large variety of biological processes and closely associated with several human diseases. The three-dimensional structure of human apo-(i.e. calcium free)-S100A1 protein was determined by NMR spectroscopy (PDB 2L0P) and its backbone dynamics established by ¹5N magnetic relaxation. Comparison of these results with the structure and backbone dynamics previously determined for bovine apo-S100A1 protein modified by disulfide formation with ß-mercaptoethanol at Cys 85 revealed that the secondary structure of both these proteins was almost identical, whereas the global structure of the latter was much more mobile than that of human apo-S100 protein. Differences between the structures of human and rat apo-S100A1 are also discussed.

Structural and motional changes induced in apo-S100A1 protein by the disulfide formation between its Cys 85 residue and beta-mercaptoethanol.

Recently, we have shown (Goch, G., Vdovenko, S., Kozlowska, H., and Bierzynski, A. (2005) FEBS J. 272, 2557-2565) that the chemical modification of Cys 85 residue of S100A1 protein by disulfide bond formation with small thiols such as glutathione, cysteine, or beta-mercaptoethanol (betaME) leads to a dramatic increase of the protein affinity for calcium. Therefore, the biological function of S100A1 as a calcium signal transmitter is probably regulated by the redox potential within the cell. Systematic, structural studies of various mixed disulfides of S100A1 in the apo and holo states are necessary to elucidate the mechanism of this phenomenon. Using NMR methods we have determined the structure of apo-S100A1-betaME and, on the basis of 15N nuclear magnetic relaxation data, we have characterized the structural dynamics of both: modified and unmodified molecules of apo-S100A1. The following effects of betaME modification have been observed: (1) Helices IV and IV' of two protein subunits are elongated by five residues (85-89). (2) Conformation of the calcium binding N-terminal loops is dramatically changed, and structural flexibility of the N-loops as well as C-loops markedly increases. (3) The angle between helices I and IV increases by approximately 20 degrees and between helices IV and IV' decreases by approximately 35 degrees . All these observations lead to the conclusion that betaME modification of apo-S100A1 makes its structure more similar to that of holo-S100A1, so that it becomes much better adjusted for calcium coordination.

Free energy of helix propagation in short polyalanine chains determined from peptide growth simulations of La3+-binding model peptides. Comparison with experimental data.

Molecular dynamics (MD) is, at present, a unique tool making it possible to study, at the atomic level, conformational transitions in peptides and proteins. Nevertheless, because MD calculations are always based on a more or less approximate physical model, using a set of approximate parameters, their reliability must be tested by comparison with experimental data. Unfortunately, it is very difficult to find a peptide system in which conformational transitions can be studied both experimentally and using MD simulations so that a direct comparison of the results obtained in both ways could be made. Such a system, containing a rigid alpha-helix nucleus stabilized by La(3+) coordination to a 12-residue sequence taken from an EF-hand protein has recently been used to determine experimentally the helix propagation parameters in very short polyalanine segments (Goch et al. (2003) Biochemistry 42: 6840-6847). The same parameters were calculated here for the same peptide system using the peptide growth simulation method with, alternatively, charmm 22 and cedar potential energy functions. The calculated free energies of the helix-coil transition are about two times too large for cedar and even three times too large for charmm 22, as compared with the experimental values. We suggest that these discrepancies have their origin in the incorrect representation of unfolded peptide backbone in solution by the molecular mechanics force fields.

Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation.

S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two alpha subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca(2+)-titration curves of Trp90 fluorescence for both the native protein and its Glu32 --> Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 --> Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K(1) =4 +/- 2 x 10(3) m(-1) (C-loops) and K(2) approximately 10(2) m(-1) (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid - a fluorescent probe of protein-surface hydrophobicity. S-Glutathionylation of the single cysteine residue (85) of the alpha subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous - four orders of magnitude - increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding to DeltaDeltaG = -6 +/- 1 kcal.mol(-1). A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.

Experimental investigation of initial steps of helix propagation in model peptides.

It is not certain whether the helix propagation parameters s(n)() (i.e., the equilibrium constants between (n - 1)- and n-residue long alpha-helices) determined from numerous studies of rather long model peptides are applicable for description of the initial steps of the helix formation during the protein folding process. From fluorescence, NMR, and calorimetric studies of a series of model peptides, containing the La(3+)-binding sequence nucleating the helix (Siedlecka, M., Goch, G., Ejchart, A., Sticht, H., and Bierzynski, A. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 903-908), we have determined, at 25 degrees C, the average values of the enthalpy DeltaH(n)() and of the helix growth parameters s(n)() describing the first four steps of helix propagation in polyalanine. The absolute values of the C-cap parameters, describing the contribution of the C-terminal residues to the helix free energy, have also been estimated for alanine (1.2 +/- 0.5) and NH(2) group (1.6 +/- 0.7). The initial four steps of the helix growth in polyalanine can be described by a common propagation parameter s = 1.54 +/- 0.04. The enthalpy DeltaH(n)() is also constant and equals -980 +/- 100 cal mol(-)(1).

Atomic resolution structure of squash trypsin inhibitor: unexpected metal coordination.

CMTI-I, a small-protein trypsin inhibitor, has been crystallized as a 4:1 protein-zinc complex. The metal is coordinated in a symmetric tetrahedral fashion by glutamate/glutamic acid side chains. The structure was solved by direct methods in the absence of prior knowledge of the special position metal centre and refined with anisotropic displacement parameters using diffraction data extending to 1.03 A. In the final calculations, the main-chain atoms of low B(eq) values were refined without restraint control. The two molecules in the asymmetric unit have a conformation that is very similar to that reported earlier for CMTI-I in complex with trypsin, despite the Met8Leu mutation of the present variant. The only significant differences are in the enzyme-binding epitope (including the Arg5 residue) and in a higher mobility loop around Glu24. The present crystal structure contains organic solvent molecules (glycerol, MPD) that interact with the inhibitor molecules in an area that is at the enzyme-inhibitor interface in the CMTI-I-trypsin complex. A perfectly ordered residue (Ala18) has an unusual Ramachandran conformation as a result of geometrical strain introduced by the three disulfide bridges that clamp the protein fold. The results confirm deficiencies of some stereochemical restraints, such as peptide planarity or the N-C(alpha)-C angle, and suggest a link between their violations and hydrogen bonding.