1,721,002 research outputs found
Calcium sensor proteins in health and disease and their potential use in nanomedicine
Full text linkProteins are biomolecules involved in virtually every process occurring in cells, characterized by a sequence of amino acids, which confer them peculiar structural features implying specific functions. Ca2+-sensor proteins are a family of proteins whose function is determined by structural changes occurring upon Ca2+-binding, taking part in a wide range of physiological and pathological processes, among which muscle contraction and phototransduction. Unraveling the complex machinery behind these physiological processes is fundamental not only for the sake of expanding our knowledge, but also to understand which mechanisms are altered in inherited pathological conditions, in order to develop potential therapeutic approaches that may help relieving or treating these disorders. Protein therapy is one of the most promising potential treatments, allowing for the substitution of dysfunctional protein pool with physiological variants. Here, a collection of biochemical and biophysical studies focused on the physiological and pathological aspects of some Ca2+-sensor proteins is presented, together with possible therapeutic implications in nanomedicine using CaF2 nanoparticles (NP) as protein carriers. The combination of experimental and computational techniques revealed itself a complementary and exhaustive approach, allowing for a multiscale investigation of protein structural and functional properties. Indeed, the high-resolution structural information provided by Molecular Dynamics (MD) simulations and Protein Structure Network (PSN) analysis can be proficiently integrated into the biophysical and biochemical experimental framework constituted by Circular Dichroism (CD) and fluorescence spectroscopy, Dynamic Light Scattering (DLS), Ca2+-binding assays and enzymatic assays. Such integrated approaches allowed us to discover that mutations of the Neuronal Calcium Sensor (NCS) protein Guanylate Cyclase Activating Protein 1 (GCAP1) associated to retinal dystrophies did not necessarily altered protein Ca2+-affinity, but rather that the pathological dysregulation of the target enzyme Guanylate Cyclase (GC) depended on more complex mechanisms, probably involving modified intramolecular communication. Moreover, PSN analysis of MD trajectories of GCAP1 highlighted that small conformational variations may strongly impact protein functionality, as the switch between activator and inhibiting states occurs through minor structural rearrangements subsequent to the Ca2+/Mg2+ exchange in specific binding sites. This suggested again that the investigation of intramolecular communication may be the key to clarify unknown complex mechanisms of not only of Ca2+-sensor proteins, but also of proteins belonging to different superfamilies
Allosteric communication pathways routed by Ca(2+)/Mg(2+) exchange in GCAP1 selectively switch target regulation modes
Full text linkGCAP1 is a neuronal calcium sensor protein that regulates the phototransduction cascade in vertebrates by switching between activator and inhibitor of the target guanylate cyclase (GC) in a Ca(2+)-dependent manner. We carried out exhaustive molecular dynamics simulations of GCAP1 and determined the intramolecular communication pathways involved in the specific GC activator/inhibitor switch. The switch was found to depend on the Mg(2+)/Ca(2+) loading states of the three EF hands and on the way the information is transferred from each EF hand to specific residues at the GCAP1/GC interface. Post-translational myristoylation is fundamental to mediate long range allosteric interactions including the EF2-EF4 coupling and the communication between EF4 and the GC binding interface. Some hubs in the identified protein network are the target of retinal dystrophy mutations, suggesting that the lack of complete inhibition of GC observed in many cases is likely due to the perturbation of intra/intermolecular communication routes
Ionic displacement of Ca2+ by Pb2+ in calmodulin is affected by arrhythmia-associated mutations
Full text linkLead is a highly toxic metal that severely perturbs physiological processes even at sub-micromolar levels, often by disrupting the Ca2+ signaling pathways. Recently, Pb2+-associated cardiac toxicity has emerged, with potential involvement of both the ubiquitous Ca2+ sensor protein calmodulin (CaM) and ryanodine receptors. In this work, we explored the hypothesis that Pb2+ contributes to the pathological phenotype of CaM variants associated with congenital arrhythmias. We performed a thorough spectroscopic and computational characterization of CaM conformational switches in the co-presence of Pb2+ and four missense mutations associated with congenital arrhythmias, namely N53I, N97S, E104A and F141L, and analyzed their effects on the recognition of a target peptide of RyR2. When bound to any of the CaM variants, Pb2+ is difficult to displace even under equimolar Ca2+ concentrations, thus locking all CaM variants in a specific conformation, which exhibits characteristics of coiled-coil assemblies. All arrhythmia-associated variants appear to be more susceptible to Pb2+ than WT CaM, as the conformational transition towards the coiled-coil conformation occurs at lower Pb2+, regardless of the presence of Ca2+, with altered cooperativity. The presence of arrhythmia-associated mutations specifically alters the cation coordination of CaM variants, in some cases involving allosteric communication between the EF-hands in the two domains. Finally, while wild type CaM increases the affinity for the RyR2 target in the presence of Pb2+, no specific pattern could be detected for all other variants, ruling out a synergistic effect of Pb2+ and mutations in the recognition process
Data_Sheet_1_Evolutionary-Conserved Allosteric Properties of Three Neuronal Calcium Sensor Proteins.pdf
Full text linkNeuronal Calcium Sensors (NCS) are highly conserved proteins specifically expressed in neurons. Calcium (Ca2+)-binding to their EF-hand motifs results in a conformational change, which is crucial for the recognition of a specific target and the downstream biological process. Here we present a comprehensive analysis of the allosteric communication between Ca2+-binding sites and the target interfaces of three NCS, namely NCS1, recoverin (Rec), and GCAP1. In particular, Rec was investigated in different Ca2+-loading states and in complex with a peptide from the Rhodopsin Kinase (GRK1) while NCS1 was studied in a Ca2+-loaded state in complex with either the same GRK1 target or a peptide from the D2 Dopamine receptor. A Protein Structure Network (PSN) accounting for persistent non-covalent interactions between amino acids was built for each protein state based on exhaustive Molecular Dynamics simulations. Structural network analysis helped unveiling the role of key amino acids in allosteric mechanisms and their evolutionary conservation among homologous proteins. Results for NCS1 highlighted allosteric inter-domain interactions between Ca2+-binding motifs and residues involved in target recognition. Robust long range, allosteric protein-target interactions were found also in Rec, in particular originating from the EF3 motif. Interestingly, Tyr 86, involved the hydrophobic packing of the N-terminal domain, was found to be a key residue for both intra- and inter-molecular communication with EF3, regardless of the presence of target or Ca2+ ions. Finally, based on a comprehensive topological PSN analysis for Rec, NCS1, and GCAP1 and multiple sequence alignments with homolog proteins, we propose that an evolution-driven correlation may exist between the amino acids mediating the highest number of persistent interactions (high-degree hubs) and their conservation. Such conservation is apparently fundamental for the specific structural dynamics required in signaling events.</p
Unveiling biochemical and physiological consequences of cone dystrophy-related mutations in GCAP1
No full textPurpose
Cone dystrophies are often associated with altered levels of calcium (Ca2+) and cyclic GMP (cGMP), the second messengers operating in the phototransduction cascade in rod and cone photoreceptors. By using a multiscale approach, we investigated the biochemical and physiological effects of four pathogenic point mutations identified in the guanylate cyclase-activating protein 1 (GCAP1), leading to the amino acid substitutions E89K, D100E, L151F and G159V.
Methods
Structure-function relationships were studied by biophysical methods, including circular dichroism to monitor secondary and tertiary structural changes in GCAP1 variants upon binding of Ca2+ and isothermal titration calorimetry to monitor the thermodynamics of Ca2+-binding. Experimental parameters describing the regulation of the target enzyme guanylate cyclase 1 (GC) by each GCAP1 variant were incorporated into a comprehensive kinetic model of phototransduction, in order to assess the effect of each individual point mutation on the whole cell response.
Results
Wild type and cone dystrophy-related point mutations in GCAP1 showed large differences in Ca2+-binding and GC regulation but, except for E89K, the structural effects of all the tested mutations are minor and involve mostly a slight rearrangement of aromatic residues in the Ca2+-bound form. System-level modeling suggests that the main effect of all point mutations on the photoresponse kinetics is a perturbation of the photocurrent shape consisting in increased amplitude and prolonged duration. However, the effect is strongly dependent on the expression levels of pathogenic GCAP1 forms as compared to the wild-type form.
Conclusion
Our data suggest that a multiscale approach combining biochemistry, biophysics and systems biology strategies allows a deep molecular understanding of dysfunctional states in photoreceptors in cone-dystrophy conditions. In particular, we conclude that the contribution of GCAP1 to the dynamic synthesis of cGMP in rod cells depends on the expression level of the wild type form, and in the case of high expression levels of cone-dystrophy GCAP1 mutants it would not contribute at all to shaping the cGMP rate, which becomes dynamically regulated solely by the other present Ca2+-sensor GCAP2
Two retinal dystrophy-associated missense mutations in GUCA1A with distinct molecular properties result in a similar aberrant regulation of the retinal guanylate cyclase
No full textTwo recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)-activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca(2+) with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca(2+). The latter mutant also shows a significantly high affinity for Mg(2+), which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca(2+), so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca(2+)-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1-EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highest-affinity Ca(2+) binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca(2+). We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca(2+) and cyclic GMP homeostasis and result in retinal degeneration
Conformational Changes in Calcium-Sensor Proteins under Molecular Crowding Conditions.
No full textFundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca2+ sensors, which undergo conformational changes in response to oscillating intracellular Ca2+ concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca2+ -binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase-activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca2+ -sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca2+ conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size-exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein-water interface. Our study provides insight into how rather small signaling proteins that have very similar three-dimensional folding patterns differ in their Ca2+ -occupied functional state under crowding conditions
Supramolecular complexes of GCAP1: implications for inherited retinal dystrophies
Full text linkGuanylate Cyclase Activating Protein 1 (GCAP1) is a calcium sensor that regulates the enzymatic activity of retinal Guanylate Cyclase 1 (GC1) in photoreceptors in a Ca2+/Mg2+ dependent manner. While point mutations in GCAP1 have been associated with inherited retinal dystrophies (IRDs), their impact on protein dimerization or on the possible interaction with the potent GC1 inhibitor RD3 (retinal degeneration protein 3) has never been investigated. Here, we integrate exhaustive in silico investigations with biochemical assays to evaluate the effects of the p.(E111V) substitution, associated with a severe form of IRD, on GCAP1 homo- and hetero-dimerization, and demonstrate that wild type (WT) GCAP1 directly interacts with RD3. Although inducing constitutive activation in GC1, the E111V substitution only slightly affects the dimerization of GCAP1. Both WT- and E111V-GCAP1 are predominantly monomeric in the absence of the GC1 target, however E111V-GCAP1 shows a stronger tendency to be monomeric in the Ca2+-bound form, corresponding to GC1 inhibiting state. Reconstitution experiments performed in the co-presence of WT-GCAP1, E111V-GCAP1 and RD3 restored nearly physiological regulation of the GC1 enzymatic activity in terms of cGMP synthesis and Ca2+-sensitivity, suggesting new scenarios for biologics-mediated treatment of GCAP1-associated IRDs
Biophysical and biochemical characterization of two novel GCAP1 mutants associated with cone-rod dystrophy
Full text linkPurpose
Guanylate Cyclase Activating Protein 1 (GCAP1) is a Ca2+-sensor protein involved in the regulation of Guanylate Cyclase (GC) during the phototransduction cascade, which initiates the visual process. An increasing number of GCAP1 mutants has been found to be associated with degenerative retinal diseases such as cone dystrophy (COD) and cone-rod dystrophy (CORD). This study is focused on the structural and functional characterization of two novel CORD-associated GCAP1 mutants, namely I107T and L84F.
Methods
Circular dichroism spectroscopy was employed to investigate changes in protein secondary and tertiary structure and in thermal stability both in the absence and in the presence of physiological 1 mM Magnesium (Mg2+) and saturating Ca2+ concentration. Variations in hydrodynamic radius of the mutants in the aforementioned conditions was monitored by dynamic light scattering. Ca2+-binding constants were estimated by a chromophoric chelator assay. The conformational transition range upon Mg2+ or Ca2+ binding was investigated by monitoring the tryptophan fluorescence in titration experiments.
Results
I107T-GCAP1 exhibited similarities with the wild type in terms of conformational and hydrodynamic radius changes upon Mg2+ or Ca2+ binding, while its Ca2+ affinity was severely impaired and its stability was increased independently on the presence of Ca2+ . Ca2+ fluorescence titrations showed a biphasic pattern similar to the COD-associated G159V mutant. L84F-GCAP1 showed structural features significantly different from the wild type, with small differences in secondary structure but major differences in tertiary structure upon Ca2+ binding. Moreover this mutant showed higher thermal stability than the wild type particularly in the presence of Ca2+ and appeared to be oligomeric both in the presence and in the absence of Ca2+ or Mg2+.
Conclusion
Our results suggest that these two novel CORD-associated GCAP1 mutants could affect GC regulation via different processes. Indeed I107T-GCAP1 might alter the Ca2+ regulation of GC by its impaired Ca2+-sensitivity, while L84F-GCAP1 may lead to different supramolecular assemblies as a consequence of its oligomeric state
Fingerprint profile of cone dystrophy related GCAP1 mutants
Full text linkPurpose
Photoreceptor cells efficiently respond to changing light conditions on a millisecond timescale by a well-balanced interplay of two second messengers, cGMP and calcium. Calcium sensor proteins like the guanylate cyclase-activating proteins (e.g. GCAP1 and GCAP2 in mammalians) control the synthesis of cGMP in a calcium-dependent manner and in astep-by-step calcium relay mode of action. Mutations in the gene GUCA1A encoding GCAP1 correlate with human cone dystrophies and are known to cause an imbalance of the calcium and cGMP homeostasis. Here we investigate the biophysical and biochemical properties of the GCAP1 mutants E89K, D100E, L151F and G159V, which are constitutive activators of photoreceptor guanylate cyclase (GC).
Methods
GCAP1 wildtype (WT) and mutants were heterologously expressed and purified. Hydrodynamic properties and calcium-binding parameters of GCAP1 variants were investigated by dynamic light scattering, isothermal titration calorimetry and size exclusion chromatography. Calcium-induced conformational changes were monitored by surface plasmon resonance. Catalytic parameters were determined by enzymatic assays using the target guanylate cyclase.
Results
Calcium-binding studies revealed three functional EF-hand calcium-binding sites in all mutants, but two EF-hands showed a several-fold lower affinity in the mutants than in WT GCAP1. Interestingly, the EF-hand with the highest affinity remained nearly unchanged. Changes in protein conformation correlated with data from dynamic light scattering and size exclusion chromatography showing a rearrangementof the protein hydration shell and a change of the dielectric constant of the protein-water interface. All mutations decrease the catalytic efficiency in regulating the target GC.
Conclusion
Point mutations of the calcium sensor GCAP1 have strong, but differential impacts on the biophysical and biochemical properties enabling the formulation of a fingerprint profile of each mutant. Thus, we further tested the consequences of dystrophy-related mutations in a kinetic model of phototransduction, in which we can access the cGMP synthesis rate resulting from either GCAP1 or GCAP2 during a photoresponse. The computational analysis revealed that the synthesis rate controlled by GCAP1 remains at a constant level, but it would not at all contribute to the shaping of the photoresponse. The latter would prominently be regulated by GCAP2
- …
