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Apo-calmodulin, a small, mainly α, soluble protein is a calcium-dependent protein activator. It is made of two N- and C-terminal domains having a sequence homology of 70%, an identical folding but different stabilities, and is thus an interesting system for unfolding studies. The use of small angle neutron scattering (SANS) and other biophysical techniques has permitted to reveal conformational difference between native and thermal denatured states of apo-calmodulin. The results show that secondary and tertiary structures of apo-calmodulin evolve in a synchronous way, indicating the absence in the unfolding pathway of molten-globule state sufficiently stable to affect transition curves. From SANS experiments, at 85 °C, apo-calmodulin adopts a polymer chain conformation with some residual local structures. After cooling down, apo-calmodulin recovers a compact state, with a secondary structure close to the native one but with a higher radius of gyration and a different tyrosine environment. In fact on a timescale of few minutes, heat denaturation of apo-calmodulin is partially reversible, but on a time scale of hours (for SANS experiments), the long exposure to heat may lead to a non-reversibility due to some chemical perturbation of the protein. In fact, from Mass Spectrometry measurements, we got evidence of dehydration and deamidation of heated apo-calmodulin.

International audience; Lamins A and C are nuclear intermediate filament proteins expressed in most differentiated somatic cells. Previous data suggested that prelamin A, the lamin A precursor, accumulates in some lipodystrophy syndromes caused by mutations in the lamin A/C gene, and binds and inactivates the sterol regulatory element binding protein 1 (SREBP1). Here we show that, in vitro, the tail regions of prelamin A, lamin A and lamin C bind a polypeptide of SREBP1. Such interactions also occur in HeLa cells, since expression of lamin tail regions impedes nucleolar accumulation of the SREBP1 polypeptide fused to a nucleolar localization signal sequence. In addition, the tail regions of A-type lamin variants that occur in Dunnigan-type familial partial lipodystrophy of (R482W) and Hutchison Gilford progeria syndrome (∆607-656) bind to the SREBP1 polypeptide in vitro, and the corresponding FLAG-tagged full-length lamin variants co-immunoprecipitate the SREBP1 polypeptide in cells. Overexpression of wild-type A-type lamins and variants favors SREBP1 polypeptide localization at the intranuclear periphery, suggesting its sequestration. Our data support the hypothesis that variation of A-type lamin protein level and spatial organization, in particular due to disease-linked mutations, influences the sequestration of SREBP1 at the nuclear envelope and thus contributes to the regulation of SREBP1 function.

Several genes in human cells are activated by physical genotoxic agents in order to regenerate cell homeostasis. Among the pathways contributing to this response, nucleotide excision repair (NER) is unique in restoring the nucleotide sequence of the DNA molecule without generating mutations. The first step of NER is mediated by a protein complex composed of XPC, RAD23B, an ubiquitin receptor and CENTRIN 2, an EF-hand calcium binding protein. These three proteins are multifunctional and participate in other important biochemical pathways. We silenced the XPC, RAD23A or RAD23B genes in HeLa cells for a long period of time by using Epstein Barr Virus-derived plasmids carrying sequences coding for small interfering RNA. XPC silencing confirms an essential role for XPC in DNA repair and cell survival after ultraviolet light irradiation. RAD23A and RAD23B participate in DNA repair and cell survival with diverging functions. Our data also indicate that CENTRIN 2 is recruited onto nuclear damaged areas quickly after irradiation and that XPC plays an important role during its internalization into the nucleus of human cells. Furthermore, the inhibition of XPC expression correlates with a decreased amount of CENTRIN 2 transcript and protein, indicating that XPC is required for the fine tuning of CENTRIN 2 gene expression. Moreover, XPC-silenced cells present a reduced concentration of CENTRIN 2 that affects both its centrosomal and nuclear localization suggesting that XPC deficiency may indirectly slow down cell division.

Cdc31, the Saccharomyces cerevisiae centrin, is an EF-hand calcium-binding protein essential for the cell division and mRNA nuclear export. We used biophysical techniques to investigate its calcium, magnesium, and protein target binding properties as well as their conformations in solution. We show here that Cdc31 displays one Ca(2+)/Mg(2+) mixed site in the N-terminal domain and two low-affinity Ca(2+) sites in the C-terminal domain. The affinity of Cdc31 for different natural target peptides (from Kar1, Sfi1, Sac3) that we obtained by isothermal titration calorimetry shows weakly Ca(2+), but also Mg(2+) dependence. The characteristics of target surface binding were shown to be similar; we highlight that the 1-4 hydrophobic amino acid motif, in a stable amphipathic α-helix, is critical for binding. Ca(2+) and Mg(2+) binding increase the α-helix content and stabilize the structure. Analysis of small-angle X-ray scattering experiments revealed that N- and C-terminal domains are not individualized in apo-Cdc31; in contrast, they are separated in the Mg(2+) state, creating a groove in the middle of the molecule that is occupied by the target peptide in the liganded form. Consequently, Mg(2+) seems to have consequences on Cdc31's function and could be important to stimulate interactions in resting cells.

Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the similar to 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -angstrom translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C+HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand. (C) 2009 Elsevier Ltd. All rights reserved

Polyoxometalates (POMs) show promising antibacterial, antiviral (particularly anti-HIV), antitumor, and anticancer activities, but the mechanism of these potential therapeutic effects remains to be elucidated at the molecular level. The interaction between the Gd-containing tungstosilicate [Gd(beta(2)-SiW(11)O(39))(2)](13-) and human serum albumin (HSA) was studied by several techniques. Fluorescence spectroscopy showed an energy transfer between the single tryptophan residue of HSA and the POM. Circular dichroism led to the conclusion that the POM significantly altered the secondary structure of HSA. Isothermal titration calorimetry revealed an enthalpy-driven binding reaction between HSA and the POM, resulting in the formation of a 1:1 complex. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

In this paper, we analyze the scaling properties of both the radius of gyration and the surface area for EF-hand calcium binding proteins. These properties are different for two conformational subfamilies: proteins with extended and compact structures, respectively. The radius of gyration is a measure of the shape of protein, whereas its surface fractal dimension is a measure of its interatomic packing. Different scaling properties for the radius of gyration underline that these two subfamilies present different shapes whilst different scaling properties for the surface area reveal different strengths of their intermolecular forces. All these data suggest different mechanisms responsible for the global folding of proteins belonging to these two subfamilies.

The EF-hand calcium-binding proteins may exist either in an extended or a compact conformation. This conformation is sometimes correlated with the function of the calcium-binding protein. For those proteins whose structure and function are known, calcium sensors are usually extended and calcium buffers compact; hence, there is interest in predicting the form of the protein starting from its sequence. In the present study, we used two different procedures: one that already exists in the literature, the sosuidumbbell algorithm, mainly based on the charges of the two EF-hand domains, and the other comprising a novel procedure that is based on linker average hydrophilicity. The linker consists of the residues that connect the domains. The two procedures were tested on 17 known-structure calcium-binding proteins and then applied to 59 unknown-structure centrins. The sosuidumbbell algorithm yielded the correct conformations for only 15 of the known-structure proteins and predicted that all centrins should be in a closed form. The linker average hydrophilicity procedure discriminated well between all the extended and non-extended forms of the known-structure calcium-binding proteins, and its prediction concerning centrins reflected well their phylogenetic classification. The linker average hydrophilicity criterion is a simple and powerful means to discriminate between extended and non-extended forms of calcium-binding proteins. What is remarkable is that only a few residues that constitute the linker (between 2 and 20 in our tested sample of proteins) are responsible for the form of the calcium-binding protein, showing that this form is mainly governed by short-range interactions.

CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca2+-saturated form and characterized the stepwise ion binding. This paper reports the 15N nuclear relaxation rates of the Ca2+-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide HN-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca2+ binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca2+-binding proteins. Supplemental material: See www.proteinscience.org

The structures of EF-hand calcium binding proteins may be classified into two distinct groups: extended and compact structures. In this paper we studied 20 different structures of calcium binding proteins using the fractal analysis. Nine structures show extended shapes, one is semi-compact and the other 10 have compact shapes. Our study reveals different fractal characteristics for protein backbones belonging to different structural classes and these observations may be correlated to the physicochemical forces governing the protein folding.

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 pigmentousum group C protein (XPC) is involved in the first step of nucleotide excision repair, with multiple functional roles including DNA damage recognition and recruitment of the repair machinery. This human protein of 940 residues forms a strong heterotrimeric complex with Rad23B and centrin 2. The structure of XPC is actually not known, and lack of significant sequence homology with proteins from structural data bases precludes any relevant prediction. Here, we present the molecular and structural characterization of a C-terminal fragment of XPC (C-XPC: 126 residues, 815-940), which was shown to be involved in centrin 2 and TFIIH binding. C-XPC may be highly expressed in E. coli, but because of its limited solubility it was purified under 6 M urea. Using bioinformatics tools, and a combination of several experimental methods (circular dichroism, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering), we show that C-XPC has a highly flexible structure under native physiological conditions, with a propensity to form helical secondary structures. Isothermal titration calorimetry experiments show that the C-XPC fragment binds human centrin 2 with high affinity and a 1:1 stoichiometry. NMR analysis indicates that the physical interaction between C-XPC and centrin 2 induces only minor conformational changes into XPC, localized around the 17-mer segment (847-863), showed to be critically involved in the centrin binding.

The C-terminal domain of human centrin 2 (C-HsCen2) strongly binds to P1-XPC, a peptide comprising 17 amino acids with a NWKLLAKGLLIRERLKR sequence. This peptide corresponds to residues N847-R863 of XPC, a protein involved in the recognition of damaged DNA during the initial step of the nucleotide excision repair pathway. The slow internal dynamics of the protein backbone in the C-HsCen-P1-XPC complex was studied by measuring the relaxation rates of zero- and double-quantum coherences involving neighboring pairs of carbonyl 13C and amide 15N nuclei. These relaxation rates, which reflect dynamics on time scales in the range of micro- to milliseconds, vary significantly along the protein backbone. Analysis of the relaxation rates at different CaCl2 concentrations and ionic strengths shows that these slow motions are mainly affected by the binding of a Ca2+ ion to the lower-affinity EF-hand III. Moreover, we discuss the possible functional role of residues that undergo differential exchange in the formation of HsCen homodimers.

International audience; The effect of increasing concentrations of 2,2,2-trifluoroethanol (TFE) on the conformational stability of the Shiga toxin B-subunit (STxB), a bacterial homopentameric protein involved in cell-surface binding and intracellular transport, has been studied by far-, near-UV circular dichroism (CD), intrinsic fluorescence, analytical ultracentrifugation, and differential scanning calorimetry (DSC) under equilibrium conditions. Our data show that the native structure of STxB is highly perturbed by the presence of TFE. In fact, at concentrations of TFE above 20% (v/v), the native pentameric conformation of the protein is cooperatively transformed into a helix-rich monomeric and partially folded conformational state with no significant tertiary structure. Additionally, no cooperative transition was detected upon a further increase in the TFE concentration (above 40% (v/v)). The thermal stability of STxB was investigated at several different TFE concentrations using DSC and CD spectroscopy. Thermal transitions at TFE concentrations of up to 20% (v/v) were successfully fitted to the two-state folding/unfolding coupled to oligomerization model consistent with the transition between a pentameric folded conformation to a monomeric state of the protein, which the presence of TFE stabilizes as a partially folded conformation.

Human centrin 2 is a component of the nucleotide excision repair system, as a subunit of the heterotrimer including xeroderma pigmentosum group C protein (XPC) and hHR23B. The C-terminal domain of centrin (C-HsCen2) binds strongly a peptide from the XPC protein (P1-XPC: N(847)-R(863)). Here, we characterize the solution Ca(2+)-dependent structural and molecular features of the C-HsCen2 in complex with P1-XPC, mainly using NMR spectroscopy and molecular modeling. The N-terminal half of the peptide, organized as an alpha helix is anchored into a deep hydrophobic cavity of the protein, because of three bulky hydrophobic residues in position 1-4-8 and electrostatic contacts with the centrin helix E. Investigation of the whole centrin interactions shows that the N-terminal domain of the protein is not involved in the complex formation and is structurally independent from the peptide-bound C-terminal domain. The complex may exist in three different binding conformations corresponding to zero, one, and two Ca(2+)-bound states, which may exchange with various rates and have distinct structural stability. The various features of the intermolecular interaction presented here constitute a centrin-specific mode for the target binding.

The solution structure of Nereis diversicolor sarcoplasmic calcium-binding protein (NSCP) in the calcium-bound form was determined by NMR spectroscopy, distance geometry and simulated annealing. Based on 1859 NOE restraints and 262 angular restraints, 17 structures were generated with a rmsd of 0.87 A from the mean structure. The solution structure, which is highly similar to the structure obtained by X-ray crystallography, includes two open EF-hand domains, which are in close contact through their hydrophobic surfaces. The internal dynamics of the protein backbone were determined by studying amide hydrogen/deuterium exchange rates and 15N nuclear relaxation. The two methods revealed a highly compact and rigid structure, with greatly restricted mobility at the two termini. For most of the amide protons, the free energy of exchange-compatible structural opening is similar to the free energy of structural stability, suggesting that isotope exchange of these protons takes place through global unfolding of the protein. Enhanced conformational flexibility was noted in the unoccupied Ca2+-binding site II, as well as the neighbouring helices. Analysis of the experimental nuclear relaxation and the molecular dynamics simulations give very similar profiles for the backbone generalized order parameter (S2), a parameter related to the amplitude of fast (picosecond to nanosecond) movements of NH-H vectors. We also noted a significant correlation between this parameter, the exchange rate, and the crystallographic B factor along the sequence.

There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography. Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca(2+)/Mg(2+) binding site of high affinity (K(d) = 3 and 10 microM for Ca(2+) and Mg(2+), respectively) and two Ca(2+)-specific sites of low affinity (K(d) = 140 microM). The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21-112. Similarly, controlled trypsinolysis on Ca(2+)-bound HsCen3 yielded a mixture of segments of residues 1-124 and 1-125. The Ca(2+)/Mg(2+) site could be assigned to this segment and thus resides in the N-terminal half of HsCen3. Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg(2+) form and a hydrophobic exposed surface for the Ca(2+) form. Thus, HsCen3 could be classified as a Ca(2+) sensor protein. In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca(2+) dependence.

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.

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer. Only the longer C-terminal domain, in the Ca(2+)-saturated state and in the presence of Na(+) ions, was amenable to structure determination by nuclear magnetic resonance. The solution structure of LC-HsCen2 reveals an open two EF-hand structure, similar to the conformation of related Ca(2+)-saturated regulatory domains. Unexpectedly, the N-terminal helix segment (F86-T94) lies over the exposed hydrophobic cavity. This unusual intramolecular interaction increases considerably the Ca(2+) affinity and constitutes a useful model for the target binding.

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.

CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes.

Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) 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 < Mg(2+) < Ca(2+). 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 Ca(2+)/Mg(2+)-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.

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.

Centrin and calmodulin (CaM) are closely related four-EF-hand Ca(2+)-binding proteins. While CaM is monomeric, centrin 2 is dimeric and binds only two Ca(2+) per dimer, likely to site IV in each monomer. Ca(2+) binding to centrin 2 displays pronounced negative cooperativity and a [Ca(2+)](0.5) of 30 microM. As in CaM, Ca(2+) binding leads to the exposure of a hydrophobic probe-accessible patch on the surface of centrin 2. Provided Ca(2+) 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 Ca(2+). Our data point to surprising differences in the mode of activation of these homologous proteins.

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by alpha-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double-labeled (13C and 15N) samples and double- and triple-resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cbeta resonances for more than 40% of the side chains in the free protein. Analysis of a 15N-labeled enzyme in complex with the bi-substrate analogue [P1,P5-bis(5'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short-range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel beta-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the 1Halpha chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. 1H/2H exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.

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 degrees 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.

The synthesis of [1,3,6,7-15N, 8-13C] adenine, using [1,3,7-15N, 8-13C]xanthine as starting labelled material, is presented. The experimental procedure is an adaptation of the synthesis methods for the corresponding unlabelled compounds. Copyright © 1999 John Wiley & Sons, Ltd.

An artificial hemoglobin-like domain has been constructed by engineering the gene coding for the multi-domain flavohemoprotein from the bacterium Erwinia chrysanthemi. This domain was designed by molecular modelling, cloned and over-expressed in Escherichia coli. The holo-protein was obtained in large quantities after extraction from inclusion bodies and refolding in presence of alkaline hemin. The purified 140-residue domain was studied and characterized to gain new insights into the biochemical function of the recombinant domain and the biological role of this new flavohemoprotein. The structural and functional features of this domain in solution were studied using far-ultraviolet circular dichroism, resonance Raman, proton-NMR spectroscopy, flash laser photolysis and molecular modelling. The recombinant domain is shown to be folded properly and active. This hemoglobin-like domain is able to bind oxygen and carbon monoxide with very high affinity. It exhibits a rapid auto-oxidation which may explain its tight association with a flavin containing reductase domain. A functional model of this hemoglobin is discussed and compared with the X-ray structures of other hemoproteins.

Phosphoglycerate kinase (PGK) is a X-linked enzyme that plays a key role in the glycolytic pathway. Twelve different variants have already been reported. We describe a new PGK variant, PGK Herlev (Asp 285-->Val), in a 69-year-old Danish patient with isolated chronic hemolysis but who had no neurological or muscular disorders. The description of the mutation is based upon PCR amplification of specific regions of the PGK gene, followed by direct sequencing. Although observed in a male patient, this mutated X-linked gene is expressed partially, i.e., both normal and substituted nucleotides are present at the same position in a ratio of approximately 1:9. The most likely explanation for this observation is based on the occurrence of a somatic mutation of the PGK gene. The relationship of structure to function in PGK Herlev, as well as in all known variants, was examined by the use of a computer model based on the known spatial structure of the yeast and horse enzymes. Such an approach can be generalized to any other protein that has been crystallized and for which x-ray diffraction data are available in a species closely related to man.

Centrins are highly conserved calcium-binding proteins involved in the nucleotide-excision repair pathway as a subunit of the heterotrimer including the XPC and hHR23B proteins. A complex formed by a Ca2+-bound human centrin 2 construct (the wild type lacking the first 25 amino acids) with a 17-mer peptide derived from the XPC sequence (residues Asn847-Arg863) was crystallized. Data were collected to 1.65 angstroms resolution from crystals grown in 30% monomethyl polyethylene glycol (MPEG) 500, 100 mM NaCl and 100 mM Bicine pH 9.0. Crystals are monoclinic and belong to space group C2, with two molecules in the asymmetric unit. The unit-cell parameters are a = 60.28, b = 59.42, c = 105.14 angstroms, alpha = gamma = 90, beta = 94.67 degrees. A heavy-atom derivative was obtained by co-crystallization with Sr2+. The substitution was rationalized by calorimetry experiments, which indicate a binding constant for Sr2+ of 4.0 x 10(4) M(-1).

FKBP59, a 59-kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non-DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi-domain organization, the N-terminal domain having a great similarity to human FKBP12 (12-kDa FK506-binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis-trans-isomerase activity, both properties being localized in the N-terminal domain (FKBP59-I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N-labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence-specific assignment of the 1H and 15N resonances was achieved using two-dimensional and three-dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from C alpha H chemical shift distribution along the sequence, the short-range and medium-range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six beta-strands and a three-turn alpha-helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The beta-sheet structure, derived from long-range connectivities between backbone protons, consists of six beta-strands defined as follows: B1, V22-I24; B2, V32-K37; B3, D50-L61; B4, T64-S68 and F76-L80; B5, E100-K107; B6, L127-F137. They are organized in an antiparallel beta-sheet with the connecting topology +1, +3, +1, -3, +1. The alpha-helix connects strand B4 to strand B5. Globally, the structure of FKBP59-I, derived from the present work, is similar to the NMR-derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The beta-sheet of the FKBP59 domain has an additional strand at the N-terminal and the alpha-helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X-ray or NMR structures of complexed FKBP12 but not in the NMR-derived, uncomplexed structure.

GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (>99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.

Yeast Dre2 is an essential Fe-S cluster-containing protein that has been implicated in cytosolic Fe-S protein biogenesis and in cell death regulation in response to oxidative stress. Its absence in yeast can be complemented by the human homologous antiapoptotic protein cytokine-induced apoptosis inhibitor 1 (also known as anamorsin), suggesting at least one common function. Using complementary techniques, we have investigated the biochemical and biophysical properties of Dre2. We show that it contains an N-terminal domain whose structure in solution consists of a stable well-structured monomer with an overall typical S-adenosylmethionine methyltransferase fold lacking two α-helices and a β-strand. The highly conserved C-terminus of Dre2, containing two Fe-S clusters, influences the flexibility of the N-terminal domain. We discuss the hypotheses that the activity of the N-terminal domain could be modulated by the redox activity of Fe-S clusters containing the C-terminus domain in vivo. Database Structural data have been deposited in the Protein Data Bank under accession number 2KM1.

To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.

Background Disrupting protein-protein interactions by small organic molecules is nowadays a promising strategy employed to block protein targets involved in different pathologies. However, structural changes occurring at the binding interfaces make difficult drug discovery processes using structure-based drug design/virtual screening approaches. Here we focused on two homologous calcium binding proteins, calmodulin and human centrin 2, involved in different cellular functions via protein-protein interactions, and known to undergo important conformational changes upon ligand binding. Results In order to find suitable protein conformations of calmodulin and centrin for further structure-based drug design/virtual screening, we performed in silico structural/energetic analysis and molecular docking of terphenyl (a mimicking alpha-helical molecule known to inhibit protein-protein interactions of calmodulin) into X-ray and NMR ensembles of calmodulin and centrin. We employed several scoring methods in order to find the best protein conformations. Our results show that docking on NMR structures of calmodulin and centrin can be very helpful to take into account conformational changes occurring at protein-protein interfaces. Conclusions NMR structures of protein-protein complexes nowadays available could efficiently be exploited for further structure-based drug design/virtual screening processes employed to design small molecule inhibitors of protein-protein interactions.

Centrins are calcium binding proteins that belong to the EF-hand (or calmodulin) superfamily, which are highly conserved among eukaryotes. Herein, we report the molecular features and binding properties of the green alga Scherffelia dubia centrin (SdCen), a member of the Chlamydomonas reinhardtii centrin (CrCen) subfamily. The Ca(2+) binding capacity of SdCen and its isolated N- and C-terminal domains (N-SdCen and C-SdCen, respectively) was investigated using flow dialysis and isothermal titration calorimetry. In contrast with human centrin 1 and 2 (from the same subfamily), but like CrCen, SdCen exhibits three physiologically significant Ca(2+) binding sites, two in the N-terminal domain and one in the C-terminal domain. Mg(2+) ions could compete with Ca(2+) in one of the N-terminal sites. When Ca(2+) binds, the N-terminal domain becomes more stable and exposes a significant hydrophobic surface that binds hydrophobic fluorescent probes. The Ca(2+) binding properties and the metal ion-induced structural changes in the C-terminal domain are comparable to those of human centrins. We used isothermal titration calorimetry to quantify the binding of SdCen, N-SdCen, and C-SdCen to three types of natural target peptides, derived from the human XPC protein (P17-XPC), the human Sfi1 protein (R17-hSfi1), and the yeast Kar1 protein (P19-Kar1). The three peptides possess the complete (P17-XPC and R17-hSfi1) or partial (P19-Kar1) centrin binding motif (W(1)L(4)L(8)). The integral SdCen exhibits two binding sites for each target peptide, with distinct affinities for each site and each peptide. The high-affinity peptide binding site corresponds to the C-terminal domain of SdCen and displays binding constants and the poor Ca(2+) sensitivities similar to those observed for human centrins. The low-affinity site constituted by the N-terminal domain is active only in the presence of Ca(2+). The thermodynamic binding parameters suggest that the C-terminal domain of SdCen may be constitutively bound to a target, while the N-terminal domain could bind a target only after a Ca(2+) signal. SdCen is also able to interact with calmodulin binding peptides (W(1)F(5)V(8)F(14) motif) with a 1:1 stoichiometry, whereas the isolated N- and C-terminal domains have a much lower affinity. These data suggest particular molecular mechanisms used by SdCen (and probably by other algal centrins) to respond to cellular Ca(2+) signals.

A supramolecular complex based on salt bridges between an artificial receptor and (Z,Z)-(BABCH), a synthetic antagonist of tissue-plasminogen activator (t-PA), leads to a notable increase of its stability in solution and a new purification route for this bioactive isomer.

Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM). The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization. Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM. The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site. The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site. In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities. This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster. The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents. Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.

In this paper we report proton two-dimensional NMR experiments on isolated alpha chains from human hemoglobin A (HbA) in the monocarboxylated state. Several J-correlated and NOE spectra in water or deuterium water and phosphate buffer (100 mM) at 310 K and pH 5.6 were acquired and analysed for the sequential assignment of the proton resonances. In addition, we used the topological data obtained from the crystal structure of alpha subunits in the monocarboxylated HbA tetramer. The assigned resonances correspond to 70% of the amino acid residues. The present results provide information on the tertiary structure of isolated alpha chains in solution, particularly in the heme region. This structure may be compared with that of the a subunits in the tetrameric HbA(CO) in crystal by comparison of observed chemical shifts and those calculated from the X-ray atomic coordinates. Overall, the global folding of the two forms are highly similar. However, this analysis points out several local conformational differences in the heme pocket and the neighboring of the unique Trp residue. Possible explanations of these differences are discussed.

A peptide of 17 amino acid residues Ac-L-K-W-K-K-L-L-K-L-L-K-K-L-L-K-L-G-NH2, designed to form an amphiphilic basic alpha-helix [DeGrado, W.F., Prendergast, F. G., Wolfe, H. R., Jr., & Cox, J. A. (1985) J. Cell. Biochem. 29, 83-93], was labeled with 15N at positions 1, 7, 9, and 10. Homo- and heteronuclear NMR techniques were used to characterize the conformational changes of the peptide when it binds to calmodulin in the presence of Ca2+ ions. The spectrum of the free peptide in aqueous solution at pH 6.3 and 298 K was completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide populates, to a significant extent, an alpha-helix conformational state, in agreement with circular dichroism measurements under similar physicochemical conditions. 15N-edited 1D spectra and 15N(omega 2)-half-filtered two-dimensional NMR experiments on the peptide in a 1:1 complex with calmodulin allowed assignment of half of the amide proton resonances and three C alpha H resonances of the bound peptide. The observed NOE connectivities between the peptide backbone protons are indicative of a stable helical secondary structure spanning at least the fragment L1-K11. The equilibrium and dynamic NMR parameters of the bound peptide are discussed in terms of a molecular interaction model.

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.

The EF-hand calcium-binding proteins may exist either in an extended or a compact conformation. This conformation is sometimes correlated with the function of the calcium-binding protein. For those proteins whose structure and function are known, calcium sensors are usually extended and calcium buffers compact; hence, there is interest in predicting the form of the protein starting from its sequence. In the present study, we used two different procedures: one that already exists in the literature, the sosuidumbbell algorithm, mainly based on the charges of the two EF-hand domains, and the other comprising a novel procedure that is based on linker average hydrophilicity. The linker consists of the residues that connect the domains. The two procedures were tested on 17 known-structure calcium-binding proteins and then applied to 59 unknown-structure centrins. The sosuidumbbell algorithm yielded the correct conformations for only 15 of the known-structure proteins and predicted that all centrins should be in a closed form. The linker average hydrophilicity procedure discriminated well between all the extended and non-extended forms of the known-structure calcium-binding proteins, and its prediction concerning centrins reflected well their phylogenetic classification. The linker average hydrophilicity criterion is a simple and powerful means to discriminate between extended and non-extended forms of calcium-binding proteins. What is remarkable is that only a few residues that constitute the linker (between 2 and 20 in our tested sample of proteins) are responsible for the form of the calcium-binding protein, showing that this form is mainly governed by short-range interactions.

The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2–PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex.

The solution conformation of a synthetic peptide of 20 amino acids (P235-254) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase was studied by proton two-dimensional NMR spectroscopy and circular dichroism. Based on the standard techniques we have assigned all the resonances in the NMR spectrum to the corresponding protons of the peptide. Analysis of the secondary chemical shift distribution and of the nuclear Overhauser effect connectivities showed no evidence for a highly populated regular conformation but suggested the tendency to form an alpha-helix around the unique Trp residue. The propensity for a helical structure is in agreement with the results of circular dichroic spectroscopy showing a slight negative band at 222 nm which was cancelled by 6 M guanidine hydrochloride. Increasing amounts of 2,2,2-trifluoroethanol (up to 40%) increase considerably the helical population of the peptide as reflected in the circular dichroic spectra. Analysis of the present results shows that the free peptide P235-254 has the tendency to form a basic amphiphilic helix. The presence of two acid residues, Glu236 and Asp239, on the hydrophilic side of the alpha-helix, which is mainly composed by basic residues, may explain the lower affinity of this peptide for calmodulin as compared with other peptides derived from calmodulin-activated enzymes.

The oxygen affinity of hemoglobin Sassari (Asp-126α aHis), a variant substituted in the α1β1 interface, was found to be 8-times greater relative to normal adult human hemoglobin. Study of the exchangeable hydrogen-bonded protons by NMR spectroscopy shows only minor changes at the α1β2 interface. In particular, the resonance previously assigned to the proton of the hydrogen bond Asp-126α1/3.Tyr-35β2 in normal hemoglobin is still present in the variant spectrum, suggesting that His-126α can also form a hydrogen bond with the Tyr-35β. The possible explanation of the increased affinity of hemoglobin Sassari and other variants substituted in the same structural region is discussed in terms of perturbations of the equilibrium between the two quaternary states.

Hemoglobin Saint Mandé (beta N102Y) is a low-affinity mutant with the substitution site situated in the quaternary-sensitive alpha 1 beta 2 interface. In adult hemoglobin the Asn102 beta contributes to the stability of the liganded (R) state, forming a hydrogen bond with Asp94 alpha. The quaternary and tertiary perturbations subsequent to the Tyr for Asn substitution in monocarboxylated hemoglobin Saint Mandé have been investigated by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis of the one-dimensional NMR spectra of the liganded and unliganded samples in 1H2O provides evidence that both R and T quaternary structures of Hb Saint Mandé are different from the corresponding ones in HbA. In the monocarboxylated form of the mutant hemoglobin, at acid pH, we have observed the disappearance of an R-type hydrogen bond and the appearance of a new one whose proton resonates like a deoxy T marker. Using two-dimensional NMR methods and on the basis of previous results on the monocarboxylated HbA, we have obtained a significant number of resonance assignments in the spectra of monocarboxylated Hb Saint Mandé at pH 5.6 in the presence or absence of a strong allosteric effector, inositol hexaphosphate. This enabled us to characterize the tertiary conformational changes (relative to the liganded normal hemoglobin) triggered by the quaternary-state modification. The observed structural variations are confined within the heme pocket regions but concern both the alpha and beta subunits. Most of them, localized in the C, F, G, and FG segments, could result directly from the side-chain substitution, while others, such as Leu141 beta, could be explained only by long-range interactions.

The molecular recognition of polyoxometalates by human serum albumin is studied using two different polyoxometalates (POMs) at pH 7.5. The results are compared with those obtained at pH 3.5 and 9.0. At pH 7.5, both POMs strongly interact with the protein with different binding behaviors. The Keggin shaped POM, [H(2)W(12)O(40)](6-) (H2W12), specifically binds the protein, forming a complex with a 1:1 stoichiometry with Ka = 2.9 x 10(6) M(-1). The binding constant decreased dramatically with the increase of the ionic strength, thus indicating a mostly electrostatic binding process. Isothermal titration calorimetry (ITC) experiments show that the binding is an enthalpically driven exothermic process. For the wheel shaped POM [NaP(5)W(30)O(110)](14-) (P5W30), there are up to five binding sites on the protein. Increasing the ionic strength changes the binding behavior significantly, leading to a simple exothermic process, with several binding sites. Competitive binding experiments indicate that the two POMs share one common binding site. In addition, they show the existence of another important binding site for P5W30. The two POMs exhibit different binding dependences on the pH. The combination of the experimental results with the knowledge of the surface map of the protein in its N-B conformation transition domain leads to the proposal for the probable binding site of POMs. The present work reveals a protein conformation change upon P5W30 binding, a new feature not explicitly documented in previous studies.

International audience; Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.

As a step toward the elucidation of the mechanistic pathways governing the known bioactivity of polyoxometalates (POMs), two representative molecules of this class of chemicals, the wheel-shaped [NaP(5)W(30)O(110)]14- (P(5)W(30)) and the Keggin-type anion [H(2)W(12)O(40)]6- (H(2)W(12)), are shown, by two independent techniques, to interact with the fatty-acid-free human serum albumin (HSA). The excited-state lifetime of the single tryptophan molecule of this protein is dramatically decreased by the binding. The quenching mechanism is found to constitute the first example of energy transfer between HSA and POMs. Such molecular recognition is believed to be a key step for subsequent evolution of the systems. Circular dichroism (CD) was used to assess the structural effects of POM binding on HSA and to confirm the interaction revealed by fluorescence studies. CD experiments showed that the two POMs have different effects on the secondary structure of the protein. Binding P(5)W(30) partially unfolds the protein whereas H(2)W(12) has no remarkable effect on the structure of the protein.