Examination of the NMR 15N chemical shifts of a number of EF-hand proteins shows that the shift value for the amido nitrogen of the residue in position 8 of a canonical EF-hand loop (or position 10 of a pseudo EF-hand loop) provides a good indication of metal occupation of that site. The NH of the residue in position 8 is covalently bonded to the carbonyl of residue 7, the only backbone carbonyl that coordinates to the metal ion in a canonical EF-hand loop. Upon metal coordination to this carbonyl, there is an appreciable deshielding of the 15N nucleus at position 8 (+4 to +8 ppm) due to the polarization of the O(7)=C(7)-N(8) amido group and the corresponding reduction in the electron density of the nitrogen atom. This deshielding effect is effectively independent of the binding of metal to the other site of an EF-hand pair, allowing the 15N shifts to be used as probes for site-specific occupancy of metal binding sites. In addition, a Ca2+-induced change in side-chain Halpha-Calpha-Cbeta-Hbeta torsion angle for isoleucine or valine residues in position 8 can also contribute to the deshielding of the amide 15N nucleus. This conformational effect occurs only in sites I or III and takes place upon binding a Ca2+ ion to the other site of an EF-hand pair (site II or IV) regardless of whether the first site is occupied. The magnitude of this effect is in the range +5 to +7 ppm. A Ca2+ titration of 15N-labeled apo-calmodulin was performed using 2D 1H-15N HSQC NMR spectra. The changes in the 15N chemical shifts and intensities for the peaks corresponding to the NH groups of residues in position 8 of the EF-hand loops allowed the amount of metal bound at sites II, III and IV to be monitored directly at partial degrees of saturation. The peak corresponding to site I could only be monitored at the beginning and end of the titration because of line broadening effects in the intermediate region of the titration. Sites III and IV both titrate preferentially and the results demonstrate clearly that sites in either domain fill effectively in parallel, consistent with a significant positive intradomain cooperativity of calcium binding.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. Previous studies have implicated mGlu5 in the pathogenesis of the disease, and many agents that target the underlying pathophysiology of FXS have focused on mGluR5 modulation. In the present work, a novel pharmacological approach for FXS is investigated. NNZ-2566, a synthetic analog of a naturally occurring neurotrophic peptide derived from insulin-like growth factor-1 (IGF-1), was administered to fmr1 knockout mice correcting learning and memory deficits, abnormal hyperactivity and social interaction, normalizing aberrant dendritic spine density, overactive ERK and Akt signaling, and macroorchidism. Altogether, our results indicate a unique disease-modifying potential for NNZ-2566 in FXS. Most importantly, the present data implicate the IGF-1 molecular pathway in the pathogenesis of FXS. A clinical trial is under way to ascertain whether these findings translate into clinical effects in FXS patients.

The fold of calmodulin (CaM) consists of two globular domains connected by a helical segment (the linker), whose conformational properties play a crucial role for the protein's molecular recognition processes. Here we investigate the structural properties of the linker by performing a 11.5 ns molecular dynamics (MD) simulation of calcium-loaded human CaM in aqueous solution. The calculations are based on the AMBER force field. The calculated S2 order parameters are in good accord with NMR data: The structure of the linker in our simulations is much more flexible than that emerging from the Homo sapiens X-ray structure, consistently with the helix unwinding observed experimentally in solution. This process occurs spontaneously in a nanosecond timescale, as observed also in a very recent simulation based on the GROMOS force field. A detailed description of the mechanism that determines the linker unwinding is provided, in which electrostatic contacts between the two globular domains play a critical role. The orientation of the domains emerging from our MD calculations is consistent both with former X-ray scattering data and a recent NMR work. Based on our findings, a rationale for the experimentally measured entropy cost associated to binding to the protein's cellular partners is also given.

Pineal hormone melatonin (N-acetyl-5-methoxytryptamine) is thought to modulate the calcium/calmodulin signaling pathway either by changing intracellular Ca 2+ concentration via activation of its G-proteincoupled membrane receptors, or through a direct interaction with calmodulin (CaM). The present work studies the direct interaction of melatonin with intact calcium-saturated CaM both experimentally, by fluorescence and nuclear magnetic resonance spectroscopies, and theoretically, by molecular dynamics simulations. The analysis of the experimental data shows that the interaction is calcium-dependent. The affinity, as obtained from monitoring 15 N and 1 H chemical shift changes for a melatonin titration, is weak (in the millimolar range) and comparable for the N-and C-terminal domains. Partial replacement of diamagnetic Ca 2+ by paramagnetic Tb 3+ allowed the measurement of interdomain NMR pseudocontact shifts and residual dipolar couplings, indicating that each domain movement in the complex is not correlated with the other one. Molecular dynamics simulations allow us to follow the dynamics of melatonin in the binding pocket of CaM. Overall, this study provides an example of how a combination of experimental and theoretical approaches can shed light on a weakly interacting system of biological and pharmacological significance.Keywords: melatonin; calmodulin; molecular dynamics; NMR; fluorescence; weak interactions Supplemental material: see www.proteinscience.org Calmodulin (CaM) is one of the most abundant, ubiquitous, and conserved proteins in eukaryotic biology. Among vertebrates, the amino acid sequence of CaM appears to be completely invariant (Hoeflich and Ikura 2002). Higher eukaryotes, including humans, possess three distinct bona fide CaM genes differentially regulated but which encode identical proteins (Hickie et al. 1983;Nojima 1989). The structure of CaM has a dumbbell shape, with two homologous Ca 2+ -binding domains linked together by a flexible tether (Barbato et al. 1992). CaM is known to interact with a large number of proteins important for Ca 2+ -dependent intracellular signaling, thus enabling the cell to control biological processes as diverse as muscle contraction, fertilization, cell Reprint requests to: Vincenzo Martorana, CNR-IBF, Istituto di Biofisica (Palermo), via U. La Malfa 153, I-90147, Palermo, Italy; e-mail: vincenzo.martorana@pa.ibf.cnr.it; fax: +390916809349.Abbreviations: CaM, calmodulin; C-CaM, C-domain of calmodulin; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; TFP, trifluoperazine; J-8, N-(8-aminooctyl)-5-iodonaphthalene-1-sulfonamide; W-7, N-(6-aminhexyl)-5-chloro-1-naphthalenesulfonamide; AAA, N-(3, 3-diphenylpropyl)-NЈ-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine; Mel, melatonin; MD, molecular dynamics; FEP, free energy perturbation; SM0, MD run of isolated Mel; SM1, MD run of Mel in solution; SC1, MD run of C-CaM in solution; SMC1, MD ...

This work shows that the partial replacement of diamagnetic Ca 2+ by paramagnetic Tb 3+ in Ca 2+ /calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb 3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca 2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb 3+ ions bound to the other domain at distances of over 40 A î from the Tb 3+ ion, shifting the resonances for these nuclei. In addition, the Tb 3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15 N-1 H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb 3+ ions. Pseudocontact shifts for NH groups and 1 H-15 N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.z 1999 Federation of European Biochemical Societies.