Identification of calcium binding sites on calsequestrin 1 and their implications for polymerization

Abstract

Biophysical studies have shown that each molecule of calsequestrin 1 (CASQ1) can bind about 70-80 Ca(2+) ions. However, the nature of Ca(2+)-binding sites has not yet been fully characterized. In this study, we employed in silico approaches to identify the Ca(2+) binding sites and to understand the molecular basis of CASQ1-Ca(2+) recognition. We built the protein model by extracting the atomic coordinates for the back-to-back dimeric unit from the recently solved hexameric CASQ1 structure (PDB id: ) and adding the missing C-terminal residues (aa350-364). Using this model we performed extensive 30 ns molecular dynamics simulations over a wide range of Ca(2+) concentrations ([Ca(2+)]). Our results show that the Ca(2+)-binding sites on CASQ1 differ both in affinity and geometry. The high affinity Ca(2+)-binding sites share a similar geometry and interestingly, the majority of them were found to be induced by increased [Ca(2+)]. We also found that the system shows maximal Ca(2+)-binding to the CAS (consecutive aspartate stretch at the C-terminus) before the rest of the CASQ1 surface becomes saturated. Simulated data show that the CASQ1 back-to-back stacking is progressively stabilized by the emergence of an increasing number of hydrophobic interactions with increasing [Ca(2+)]. Further, this study shows that the CAS domain assumes a compact structure with an increase in Ca(2+) binding, which suggests that the CAS domain might function as a Ca(2+)-sensor that may be a novel structural motif to sense metal. We propose the term "Dn-motif" for the CAS domain.

Figure 1. Changes in the surface charge pattern of CASQ1 with increasing Ca²⁺ concentrations

Surface charge was calculated for the average structure for each system indicated in the figure. Red, blue and white indicate negative, positive and neutral charge on the molecular surface. The color scale used to represent the Poisson Boltzmann electrostatic potential isosurface range is from −4 to +4 KT/e (where K - Boltzmann constant, T - temperature, and e - electron charge. CASQ1 gains electro-positivity on the surface in the 120 Ca²⁺-system, even if the surface negative charge is almost neutralized already in the 80 Ca²⁺-system.

Figure 2. Number of bound Ca²⁺ in the presence of increasing [Ca²⁺]

(A) Ca²⁺ bound to the Dn-motif (from amino acid 350 to 364 on the C-terminus) of each monomer. The Dn-motif at the dimeric interface binds less Ca²⁺ than the free Dn-motif. (B) Total number of Ca²⁺ ions bound to the rest of the protein (amino acids 1–350) surface of each monomer. Both the monomers bind almost similar number of Ca²⁺ ions although at intermediate [Ca²⁺] the monomer “chain B” binds more Ca²⁺ ions than “chain C”. Observing the slope of the curves it seems that both the monomers still possess the ability to bind even a higher number of Ca²⁺ ions, if provided.

Figure 3. Identification of high affinity Ca²⁺-binding sites independent of [Ca²⁺]

Site 1 and site 3 are representative examples of high affinity Ca²⁺ binding sites on CASQ1 that are independent of [Ca²⁺]. Site 1 (A-B) and site 3 (C-D) bind Ca²⁺ with very similar geometry and affinity (see Table 1). System with 20 Ca²⁺ ions is referred to as “Low [Ca²⁺]”, whereas System with 80 Ca²⁺ ions is referred to as “High [Ca²⁺]”. The protein residues are represented in ball-stick representation, Ca²⁺ ion in magneta and water molecules that are present within 3.0 Å of Ca²⁺ ion in light blue.

Figure 4. Identification of [Ca²⁺] dependent high affinity Ca²⁺-binding sites

Representative examples of Ca²⁺ binding sites on CASQ1 that are induced by an increase in [Ca²⁺] in the system. Site 5 (A-B) and site 7 (C-D) show progressively a more favorable Ca²⁺-binding geometry and better binding affinity increasing Ca²⁺ in the system (see Table 1).

Figure 5. [Ca²⁺] dependent intermediate affinity Ca²⁺ binding sites on CASQ1

Some Ca²⁺ binding sites on CASQ1, like site 10, are induced to bind Ca²⁺ with high affinity only at intermediate [Ca²⁺]. (A) Site 10 is constituted by residues D319, E350 and E354, that show no sensitivity to Ca²⁺ when only 20 Ca²⁺ ions are present in the system. (B) Site 10 binds Ca²⁺ when 40 Ca²⁺ ions are present in the system. (C) Site 10 recognizes Ca²⁺ and binds with very high affinity and an optimal geometry when [Ca²⁺] increases to 80 ions. (D) With further increase in [Ca²⁺] to 120 ions, we note disruption of the optimal binding topology of site 10 and thus the interaction energy drastically reduces.

Figure 6. Characterization of the inter-molecular interaction at the back-to-back dimeric interface of CASQ1

(A-C) Back-to-back stacking is stabilized by promiscuous hydrophobic interactions that increase with the increase in [Ca²⁺]. Hydrogen bonding (shown in green dotted lines, with distance in Å) does not play a major role in the back-to-back interaction.

Figure 7. The Dn-motif of CASQ1 assumes compact conformation upon binding of Ca²⁺ ions

(A-C) The CAS or the Dn-motif is observed to exist in three main conformations. The linear conformation of CAS is characterized by the presence of the least number of Ca²⁺-bound as well as by the energetically least stable state. Additional Ca²⁺-binding leads to to an intermediate conformer, which was found to be energetically more stable. Finally, the Dn-motif saturation by Ca²⁺ resulted in the most energetically stable and a very compact structure.

Figure 8. Proposed model for Ca²⁺-CASQ1 interaction and polymerization

The centre of each domain of the CASQ1 molecule is hydrophobic and must undergo hydrophobic collapse to form the domains. The core region of CASQ1 at the interface, between the three domains is acidic, which is characteristic feature of the CASQ-protein family. The inter-domain charge neutralization by Ca²⁺ is necessary for the three domains to come to close proximity to form a compact monomer. The Dn-motif of CASQ1 is composed of 13 aspartic acids that can not bind more than 8 Ca²⁺ ions, whereas CASQ1 monomer has capacity to bind 70–80 Ca²⁺ ions. Therefore, the CASQ1 structural domains can bind more than 50 Ca²⁺. Existence of sites that can switch to an high affinity sites at varying [Ca²⁺] allows CASQ1 to release Ca²⁺ without the necessity of undergoing depolymerization. This observation holds in most part of the “physiological range of Ca²⁺ variations”. Dotted lines indicate the extreme Ca²⁺ concentrations that might be important only during higher physiological Calcium demand.