Abstract
Neuronal 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.
Highlights
Calcium (Ca2+) is a universal second messenger whose changes in concentration contribute to the regulation of a variety of biological processes ranging from muscle contraction (Ebashi and Endo, 1968) to signal transduction (Berridge et al, 2003) and neuronal signaling (Augustine et al, 2003)
The structures with the highest resolution and sequence coverage available were chosen for both Rec and Neuronal Calcium Sensor 1 (NCS1), resulting in the following models: Ca2+-free Rec (“tense Recoverin” or Rec-T, Tanaka et al, 1995), Rec with one Ca2+ bound to EF3 (“intermediate state Recoverin” or Rec-I, Ames et al, 2002), Ca2+-loaded Rec (“relaxed Recoverin” or Rec-R, Ames et al, 1997), Ca2+-loaded Rec bound to GRK1 peptide (Rec-GRK1, Ames et al, 2006; Zernii et al, 2011; Ames and Lim, 2012), Ca2+-loaded uncomplexed NCS1 (“isolated NCS1” or NCS1-iso), Ca2+-loaded NCS1 bound to D2 Dopamine receptor peptides (NCS1-D2R) and Ca2+-loaded NCS1 bound to Rhodopsin Kinase peptide [NCS1-GRK1, (Bourne et al, 2001; Pandalaneni et al, 2015)]
We previously identified Guanylate Cyclase Activating Protein 1 (GCAP1) hub residues that, when mutated, were associated with retinal dystrophies (Marino and Dell’orco, 2016), namely: D100, which is the target of the D100E/G substitutions (Kitiratschky et al, 2009; Dell’orco et al, 2010; Nong et al, 2014); L84, which is the target of the L84F substitution (Kamenarova et al, 2013; Marino et al, 2015a); Y99, associated with the Y99C mutation (Payne et al, 1998; Sokal et al, 1998); E155, associated with the E155A and E155G mutations (Wilkie et al, 2001); I143, found to be mutated in I143T/N (Nishiguchi et al, 2004)
Summary
Calcium (Ca2+) is a universal second messenger whose changes in concentration contribute to the regulation of a variety of biological processes ranging from muscle contraction (Ebashi and Endo, 1968) to signal transduction (Berridge et al, 2003) and neuronal signaling (Augustine et al, 2003). Neuronal Calcium Sensor proteins (NCS) though, are a tissue-specific and highly specialized class of proteins able to regulate a limited number of targets (Burgoyne, 2007; Burgoyne and Haynes, 2015) involved in a large array of neuronal transmission processes. NCS proteins may have only one predominant biological target, as is as case for Rec and GCAP1, or even regulate the same effectors, as is the case for the regulation of Rhodopsin Kinase (GRK1) by both Rec and NCS1
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