Evolutionary-Conserved Allosteric Properties of Three Neuronal Calcium Sensor Proteins

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.

Keywords: GCAP1; NCS1; molecular dynamics; neuronal calcium sensors; protein structure network; recoverin.

Figure 1

Schematic representation of the intramolecular communication between NCS1 EF-hands and GRK1 (left) or D₂R (right) interface residues. NCS1 shape is represented in gray, helices of the four EF-hands are represented by green cylinders where the entering helix is in front of the exiting helix, GRK1 and D₂R peptides are shown in purple and yellow cylinders, respectively. Occupied Ca²⁺-binding sites are represented as red circles. For each peptide, residues representing EF2 (A,D), EF3 (B,E) and EF4 (C,F), and the two residues with highest CR (Figure S3) are shown as black dots and labeled. Interactions between interface residues and target peptides are represented by black dashed lines. The communication between EF-hand representatives and interface residues in the absence and in the presence of targets is shown, respectively, by orange and blue arrows, whose width is proportional to CR values (Figure S3).

Figure 2

Schematic representation of the intramolecular communication between Rec EF-hands and GRK1 interface residues. Rec shape is represented in gray, helices of the four EF-hands are represented by green cylinders where the entering helix is in front of the exiting helix, GRK1 is shown as a solid or transparent purple cylinder depending on its presence. Empty Ca²⁺ binding sites are represented as white circles, occupied Ca²⁺-binding sites are represented as red circles. Panels show Rec-T (apo, A), Rec-I (EF3-Ca²⁺, B), Rec-R (Ca²⁺-loaded, C) and Rec-GRK1 (Ca²⁺-loaded and peptide bound, D). For each case residues representing EF2 and EF3, namely E85 and E121, and the two residues with highest CR (Figure S4) are shown as black dots and labeled. Interactions between interface residues and GRK1 peptide are shown by black dashed lines. The communication between EF-hand representatives and interface residues is represented by dark red (E85) and teal (E121) arrows whose width is proportional to CR values (Figure S4).

Figure 3

Intermolecular communication pathways between GRK1 peptide and EF-hands of NCS1 (top panels) and Rec (bottom panels). Ca²⁺ ions are shown as red spheres, NCS1, Rec and GRK1 structures are shown in cylindrical cartoon and colored in cyan, light green and purple, respectively, Rec myristoyl group is represented in light green sticks. Pathways with the highest cumulative SB connecting EF-hand representatives and GRK1 residues with the highest CR are shown in sticks and spheres. In detail, pathway connecting NCS1 E84 and GRK1 S13 (A) is shown in dark green, pathway connecting NCS1 E120 and GRK1 N12 (B) is shown in blue, pathway connecting NCS1 E168 and GRK1 L6 (C) is shown in yellow, while pathways connecting Rec E85 and GRK1 N12 (D) and Rec E121 and GRK1 A11 (E) are shown in orange and teal, respectively. Pathway residues are labeled in black, residues shared by two pathways are labeled in red, GRK1 residues are underlined.

Figure 4

Protein Structure Networks of Ca²⁺-loaded NCS1 (left) and Rec (right) and hub identification. Protein secondary structures are shown as cyan (NCS1) and light green (Rec) cartoons, Ca²⁺ ions are shown as red spheres, Rec myristoyl group is represented in light green sticks. Non-covalent interaction (edges) between residues (nodes) is represented as gray sticks connecting residues Cα or Ca²⁺ ions. Cα atoms are shown as spheres with radius size proportional to the number of connections and colored in blue (degree 7 hubs) or orange (degree 8 hubs).

Figure 5

Multiple Sequence Alignment of Rec, NCS1, and GCAP1. Hub residues are colored according to their degree; therefore degree 8 hubs are in dark red, degree 7 hubs are in orange, degree 6 hubs are in green and degree 5 hubs are in blue. Hub residues with degree ≥ 5 in all three proteins are framed and colored in white, conserved residues are highlighted in gray, identical residues are highlighted in red.

Figure 6

Correlation between hub degree and average residue conservation in UniRef50 clusters for Rec, NCS1, and GCAP1. Average residue conservation in UniRef50 clusters is plotted against hub degree for Rec (green diamonds), NCS1 (blue circles), and GCAP1 (red squares). Residue conservation in UniRef50 clusters shown in Figure 6 and in Tables S1–S3,S5 was calculated as the ratio between the number of sequences where the residue of the seed sequence was conserved and the total number of sequences in the cluster. When a residue was a hub in different signaling states, even with a different degree, only one occurrence of the residue was considered for the calculation of the average of given hub degree. Linear regression of average residue conservation is shown as a black line.

Figure 7

Structural insights into hydrophobic interaction network involving NCS1-iso residue F85 (left) and Rec-GRK1 residue Y86 (right). Protein secondary structures are shown as cyan (NCS1), light green (Rec) and purple (GRK1) cartoons, Ca²⁺ ions are shown as red spheres. Residues F85 and Y86 are represented as red sticks and labeled, hydrophobic residues surrounding F85 or Y86 are represented as blue sticks and labeled, residue F15, belonging to GRK1 peptide is underlined.