Constantin T. Craescu

Molecular Interactions between Wells-Dawson Type Polyoxometalates and Human Serum Albumin

Binding human serum albumin (HSA) of three polyoxometalates (POMs) with the Wells-Dawson structure, alpha(2)-[P2W17O61]10- (abbreviated as alpha(2)-P2W17) and two of its metal-substituted derivatives, alpha(2)-[NiP2W17O61]8- and alpha(2)-[CuP2W17O61]8- (alpha(2)-P2W17Ni and alpha(2)-P2W17Cu, respectively) was studied in an aqueous medium at pH 7.5. Fluorescence quenching, circular dichroism (CD), thermal denaturation, and isothermal titration calorimetry (ITC) were used for this purpose. The results were compared with those obtained previously with the Keggin structure POM, [H2W12O40]6- (H2W12), and the wheel-shaped structure, [NaP5W30O110]14- (P5W30). All these POMs bind HSA mainly by electrostatic interactions. Comparison of the physical characteristics and HSA interaction parameters for the POMs of the present work and those studied previously showed that the overall charge of the clusters is not the single parameter governing the binding process and its consequences. In contrast, besides the influences of the structure, the dimension and/or weight of the POMs, the results have permitted highlighting of the importance of each POM atomic composition for its binding behavior.

Xeroderma Pigmentosum Group C Protein Possesses a High Affinity Binding Site to Human Centrin 2 and Calmodulin

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca2+-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca2+ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway. Analysis of the XPC sequence (940 residues), using a calmodulin target recognition software, enabled us to predict two putative binding sites. The binding properties of the two corresponding peptides were investigated by isothermal titration calorimetry. Only one of the peptides (P1-XPC) interacts strongly (Ka = 2.2 × 108 m-1, stoichiometry 1:1) with HsCen2 in a Ca2+-dependent manner. This peptide also binds, with a similar affinity (Ka = 1.1 × 108 m-1) to a C-terminal construct of HsCen2, indicating that the interaction with the integral protein is mainly the result of the contribution of the C-terminal half. The second peptide (P2-XPC) failed to show any detectable binding either to HsCen2 or to its C-terminal lobe. The two peptides interact with different affinities and mechanisms with calmodulin. Circular dichroism and nuclear magnetic resonance were used to structurally characterize the complex formed by the C-terminal domain of HsCen2 with P1-XPC.

Cation- and peptide-binding properties of human centrin 2

Centrin and calmodulin (CaM) are closely related four‐EF‐hand Ca2+‐binding proteins. While CaM is monomeric, centrin 2 is dimeric and binds only two Ca2+ per dimer, likely to site IV in each monomer. Ca2+ binding to centrin 2 displays pronounced negative cooperativity and a [Ca2+]0.5 of 30 μM. As in CaM, Ca2+ binding leads to the exposure of a hydrophobic probe‐accessible patch on the surface of centrin 2. Provided Ca2+ is present, centrin 2 forms a 1:1 peptide:monomer complex with melittin with an affinity of 100 nM. The complex binds four instead of two Ca2+. Our data point to surprising differences in the mode of activation of these homologous proteins.

Abnormality of phospholipid transverse diffusion in sickle erythrocytes.

We have used spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to compare the transverse diffusion rates of lipids in normal and sickle erythrocytes. The beta-chain of the spin-labeled lipids was a short chain (five carbons) providing the spin labels with a relative water solubility, and hence permitting their rapid incorporation into cell membranes. The orientation of the labeled lipids in the membranes was assayed by selective chemical reduction of the nitroxide labels embedded in the outer leaflet. We have found that all three spin-labeled phospholipids are initially incorporated in the outer leaflet. Upon incubation at 4 degrees C the aminophospholipids, not the phosphatidylcholine, diffuse toward the inner leaflet within 3 h. The transverse diffusion rate of aminophospholipids is reduced by 41% (phosphatidylserine) and 14% (phosphatidylethanolamine) in homozygote sickle cells (SS) when compared with normal cells (AA) or heterozygote cells (AS or SC). At equilibrium the asymmetric distribution of spin-labeled phospholipids resulting from this selective diffusion is also reduced in SS cells when compared with AA, SC, or AS cells. This reduced asymmetry was not found in a reticulocyte-rich blood sample (hemoglobin A), indicating that the age of the cell cannot be responsible for this phenomenon. Moreover, because at low temperatures the sickling process does not occur, the observed perturbations in phospholipid organization reflect preexisting membrane abnormalities in sickle cells. Ghosts loaded with ATP give the same results. Varying the concentration of intracellular calcium had no effect on lipid diffusion, except at very high free calcium concentrations (3 microM) when diffusion was practically abolished. We suggest that membrane protein alterations may be part of the explanation of the observed abnormalities.

Binding of human centrin 2 to the centrosomal protein hSfi1

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium‐binding EF‐hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin‐binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro‐containing human repeat. The two standard peptides bind human centrin 2 and its isolated C‐terminal domain with high affinity (∼ 107 m−1) by an enthalpy‐driven mechanism, with a moderate Ca2+ dependence. The Pro‐containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C‐terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well‐conserved Trp residue situated in the C‐terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self‐assembly, we propose a working model for the role of centrin–Sfi1 interactions in the dynamic structure of centrosome‐associated contractile fibers.

Identification of the Binding Site for the Regulatory Calcium-Binding Domain in the Catalytic Domain of NOX5

NADPH oxidases (NOX) are important superoxide producing enzymes that regulate a variety of physiological and pathological processes such as bacteria killing, angiogenesis, sperm-oocyte fusion, and oxygen sensing. NOX5 is a member of the NOX family but distinct from the others by the fact that it contains a long N-terminus with four EF-hand Ca(2+)-binding sites (NOX5-EF). NOX5 generates superoxide in response to intracellular Ca(2+) elevation in vivo and in a cell-free system. Previously, we have shown that the regulatory N-terminal EF-hand domain interacts directly and in a Ca(2+)-dependent manner with the catalytic C-terminal catalytic dehydrogenase domain (CDHD) of the enzyme, leading to its activation. Here we have characterized the interaction site for the regulatory NOX5-EF in the catalytic CDHD of NOX5 using cloned fragments and synthetic peptides of the CDHD. The interaction was monitored with pull-down techniques, cross-linking experiments, tryptophan fluorescence, hydrophobic exposure, isothermal titration calorimetry, and cell-free system enzymatic assays. This site is composed of two short segments: the 637-660 segment, referred to as the regulatory EF-hand-binding domain (REFBD), and the 489-505 segment, previously identified as the phosphorylation region (PhosR). NOX5-EF binds to these two segments in a Ca(2+)-dependent way, and the superoxide generation by NOX5 depends on this interaction. Controlled proteolysis suggests that the REFBD is autoinhibitory and inhibition is relieved by NOX5-EF.

Structural and nucleotide-binding properties of YajQ and YnaF, two Escherichia coli proteins of unknown function

Structural genomics is a new approach in functional assignment of proteins identified via whole‐genome sequencing programs. Its rationale is that nonhomologous proteins performing similar or related biological functions might have similar tertiary structure. We used dye pseudoaffinity chromatography, two‐dimensional gel electrophoresis, and mass spectrometry to identify two novel Escherichia coli nucleotide‐binding proteins, YnaF and YajQ. YnaF exhibited significant sequence identity with MJ0577, an ATP‐binding protein from a hyperthermophile (Methanococcus jannaschii), and with UspA, a protein from Haemophilus influenzae that belongs to the Universal Stress Protein family. YnaF conserves the ATP‐binding site and the dimeric structure observed in the crystal of MJ0577. The protein YajQ, present in many bacterial genomes, is missing in eukaryotes. In the absence of significant similarities of YajQ to any solved structure, we determined its structural and ligand‐binding properties by NMR and isothermal titration calorimetry. We demonstrate that YajQ is composed of two domains, each centered on a β‐sheet, that are connected by two helical segments. NMR studies, corroborated with local sequence conservation among YajQ homologs in various bacteria, indicate that one of the β‐sheets is mostly involved in biological activity.

A new subfamily of short bacterial adenylate kinases with the Mycobacterium tuberculosis enzyme as a model: A predictive and experimental study.

The adk gene from Mycobacterium tuberculosis codes for an enzyme of 181 amino acids. A sequence comparison with 52 different forms of adenylate kinases (AK) suggests that the enzyme from M. tuberculosis belongs to a new subfamily of “short” bacterial AKs. The recombinant protein, overexpressed in Escherichia coli, exhibits a low catalytic activity and an unexpectedly high thermal stability (Tm = 64.8°C). Based on various spectroscopic data, on the known three‐dimensional structure of the AK from E. coli and on secondary structure predictions for various sequenced AKs, we propose a structural model for AK from M. tuberculosis (AKmt). Proteins 1999;36:238–248.

Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule.

Nereis sarcoplasmic Ca2+‐binding protein (NSCP) is a calcium buffer protein that binds Ca2+ ions with high affinity but is also able to bind Mg2+ ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg2+ < Ca2+. The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo‐NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca2+/Mg2+‐bound states. Gel filtration experiments showed that, although the metal‐bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal‐dependent structural changes and stability are discussed. Proteins 2000;40:177–184.

Biophysical Study of Thermal Denaturation of Apo-Calmodulin: Dynamics of Native and Unfolded States

Apo-calmodulin, a small, mainly alpha, soluble protein is a calcium-dependent protein activator. This article presents a study of internal dynamics of native and thermal unfolded apo-calmodulin, using quasi-elastic neutron scattering. This technique can probe protein internal dynamics in the picosecond timescale and in the nanometer length-scale. It appears that a dynamical transition is associated with thermal denaturation of apo-calmodulin. This dynamical transition goes together with a decrease of the confinement of hydrogen atoms, a decrease of immobile protons proportion and an increase of dynamical heterogeneity. The comparison of native and unfolded states dynamics suggests that the dynamics of protein atoms is more influenced by their distance to the backbone than by their solvent exposure.