Single-Molecule Folding Mechanism of an EF-Hand Neuronal Calcium Sensor

Abstract

EF-hand calcium sensors respond structurally to changes in intracellular Ca(2+) concentration, triggering diverse cellular responses and resulting in broad interactomes. Despite impressive advances in decoding their structure-function relationships, the folding mechanism of neuronal calcium sensors is still elusive. We used single-molecule optical tweezers to study the folding mechanism of the human neuronal calcium sensor 1 (NCS1). Two intermediate structures induced by Ca(2+) binding to the EF-hands were observed during refolding. The complete folding of the C domain is obligatory for the folding of the N domain, showing striking interdomain dependence. Molecular dynamics results reveal the atomistic details of the unfolding process and rationalize the different domain stabilities during mechanical unfolding. Through constant-force experiments and hidden Markov model analysis, the free energy landscape of the protein was reconstructed. Our results emphasize that NCS1 has evolved a remarkable complex interdomain cooperativity and a fundamentally different folding mechanism compared to structurally related proteins.

Direct single-molecule observation of calcium-dependent misfolding in human neuronal calcium sensor-1

“…Because folding reaches I2 before any misfolding occurs and because previous manipulation of the EF3 binding site disables folding, with no apparent native transitions and with similar behavior to the apo form of NCS-1 (39), this rules out EF3 as the origin for generation of misfolded states. In contrast, variants carrying disabled EF2 (NCS-1 EF2 ) or EF4 (NCS-1 EF4 ) binding sites still retain some native and nonnative folding transitions (39). Importantly, NCS-1 EF2 never populates M1 or M2, as the C domain always folds properly into its native structure, even at high Ca 2+ concentrations and relaxation speeds (Fig.…”

“…Individual NCS-1 molecules were manipulated with polystyrene beads by means of DNA molecular handles covalently attached to cysteine residues engineered at positions 4 and 188, as previously reported (39) (Fig. 1B and Methods).…”

“…1B and Methods). To identify and characterize misfolding pathways of NCS-1, we first performed constant-velocity experiments, where the molecule is stretched and relaxed by moving the pipette at constant speed relative to the optical trap (16,39,40). Under these experimental conditions, we have previously shown that NCS-1 unfolds in an apparent three-state manner and folds back into its native state through a process that starts with the folding of the C domain, undergoing a major conformational change (U→I2) followed by a minor rearrangement (I2→I1), and ends with the folding of the N domain at lower forces (I1→N transition) (Fig.…”

“…We therefore separately introduced classical mutations that prevent ion binding into each of the three calcium-binding EFhand loops (39). Because folding reaches I2 before any misfolding occurs and because previous manipulation of the EF3 binding site disables folding, with no apparent native transitions and with similar behavior to the apo form of NCS-1 (39), this rules out EF3 as the origin for generation of misfolded states.…”

“…1E). In contrast to NCS-1 EF2 , NCS-1 EF4 never transits from I2 to I1, because this transition involves binding of Ca 2+ to EF4 (39), and thus its C domain never reaches its native conformation. Nonetheless and surprisingly, with NCS-1 EF4 , we could not detect any transitions from I2 to conformations resembling M1 or M2 (Fig.…”