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

Neurodegenerative disorders are strongly linked to protein misfolding, and crucial to their explication is a detailed understanding of the underlying structural rearrangements and pathways that govern the formation of misfolded states. Here we use single-molecule optical tweezers to monitor misfolding reactions of the human neuronal calcium sensor-1, a multispecific EF-hand protein involved in neurotransmitter release and linked to severe neurological diseases. We directly observed two misfolding trajectories leading to distinct kinetically trapped misfolded conformations. Both trajectories originate from an on-pathway intermediate state and compete with native folding in a calcium-dependent manner. The relative probability of the different trajectories could be affected by modulating the relaxation rate of applied force, demonstrating an unprecedented real-time control over the free-energy landscape of a protein. Constant-force experiments in combination with hidden Markov analysis revealed the free-energy landscape of the misfolding transitions under both physiological and pathological calcium concentrations. Remarkably for a calcium sensor, we found that higher calcium concentrations increased the lifetimes of the misfolded conformations, slowing productive folding to the native state. We propose a rugged, multidimensional energy landscape for neuronal calcium sensor-1 and speculate on a direct link between protein misfolding and calcium dysregulation that could play a role in neurodegeneration.Most proteins have evolved to fold rapidly into a specific and functional 3D structure immediately after translation from the ribosome. The folding process is, however, not adequately efficient to prevent the occurrence of misfolded states in vivo (1), especially in the case of larger multidomain proteins which comprise roughly 75% of the human proteome (2, 3). Normally, to tackle and destroy these unproductive structures, cells are equipped with competent clean-up machinery, such as chaperones, proteasomes, and unfoldases (4). If misfolding cannot be ameliorated, these nonnative states accumulate in the cell to form aggregates with potential pathophysiological consequences (5).The emerging view that protein misfolding is a common phenomenon in living cells is still largely unsubstantiated, as detecting and characterizing misfolded states has been experimentally challenging (2, 6). The mechanistic details that have accumulated over the last decades on misfolding have mostly come from studies on the resulting oligomeric structures and amyloid formation (1), whereas our understanding of the structural rearrangements and pathways leading to precursory misfolded states is still highly incomplete. Importantly, the formation of prefibrillar monomeric and oligomeric misfolded states is, contrary to amyloids, reversible and thus these states provide a potential target for drug design.Sparse populations and their associated weak signals limit the use of traditional bulk methods for monitoring the early events of misfolding, and relatively few systems have been studied in detail (7–13). Now, single-molecule force spectroscopy techniques, such as optical tweezers, enable detection of rare alternative folding pathways and short-lived misfolded states by direct mechanical manipulation (14–19). Although aggregation requires more than one molecule, nonnative structural rearrangements within a single molecule only report on monomeric misfolded states. Recent works have exploited these properties to study misfolding of well-known disease-related proteins, such as the prion protein, as well as proteins not generally associated with misfolding, such as the EF-hand calcium sensor calmodulin (CaM) (20–22).The EF-hand superfamily of calcium sensors is responsible for translating changing levels of intracellular Ca²⁺ concentration into a biochemical signal through conformational changes that allow them to interact with an array of binding targets (23). The subfamily of neuronal calcium sensors (NCS) is mostly expressed in neurons and currently includes 15 members (24, 25). Neuronal calcium sensor-1 (NCS-1) is the most ancient member of this family (Fig. 1A), and it has been functionally associated with cognitive processes, such as learning and memory (26, 27), and with a number of cellular processes such as neurotransmitter release (28, 29), and regulation of ion channels, and G protein coupled receptors (GPCRs) (24, 30), including the dopamine receptor D2 (31). NCS-1 has also been linked to serious neurodegenerative disorders including schizophrenia, bipolar disorder (BD) (32), and autism (33, 34). However, the dysfunctions of NCS-1 are poorly characterized on the molecular level, and whether they involve altered functional profiles or loss of function due to formation of misfolded states is currently unknown.Open in a separate windowFig. 1.Misfolding pathways of NCS-1. (A) The NMR structure of NCS-1 (PDB 2LCP), with the N domain (EF1/EF2) depicted in gray and the C domain (EF3/EF4) in blue. Black spheres represent Ca²⁺ ions. EF1 does not bind Ca²⁺ because of a conserved cysteine-proline mutation (35). (B) Sketch of the experimental setup. NCS-1 was tethered between functionalized beads via DNA handles and stretched and relaxed by moving the pipette relative to the optical trap (16, 62). (C) Native folding pathway of NCS-1. After being mechanically stretched and unfolded (red trace), NCS-1 refolds upon relaxation of the applied force into its native state via two intermediate states, I1 and I2. Dashed lines are worm-like-chain fits to the data. Color-coded arrows indicate the pulling/relaxing directions. (D) Misfolding pathways of NCS-1. During refolding, NCS-1 sometimes follows alternative pathways leading to misfolded states M1 (blue) or M2 (green), which are less compact than the native state (red). Dashed lines are worm-like-chain fits to the data. (E) Rescue of the native state of NCS-1. During relaxation (black), the molecule misfolded. During stretching (red), nonnative contacts of the misfolded conformation are progressively broken, until the molecule can find its native folding pathway (“rescue transition”). (F) Fraction of folding pathways leading to either M1 or M2 as a function of Ca²⁺ concentration and relaxation speed. Higher Ca²⁺ concentrations and relaxation speeds facilitate NCS-1 misfolding. Error bars indicate SEs of mean. At least five different molecules were used for each calcium concentration.Because only a few systems have been studied experimentally, little is known about folding and/or misfolding mechanisms of members of the EF-hand superfamily (21, 35–37). The extensively studied CaM has been shown on the single-molecule level to frequently visit misfolded states that slow down the overall folding rate of the protein (21). The physiological consequences of CaM misfolding have not yet been explored. NCS-1 shares modest sequence homology with CaM, mostly within and around the calcium binding sites (24). Similar to CaM, NCS-1 contains four EF hands organized in two EF domains (Fig. 1A) yet it exhibits a larger number of interdomain contacts (38), a feature that has been suggested to increase the probability of misfolding in proteins (2). The formation of misfolded states along the folding pathway of NCS-1 may have important consequences with regards to its function as a calcium sensor and might also play a role in disease pathologies.Using optical tweezers, we have recently characterized the native folding pathway of NCS-1 (39). Here we use a similar experimental approach to monitor individual NCS-1 molecules as they populate nonnative misfolded states in real time. We identified two misfolding trajectories leading to two distinct misfolded conformations, characterized by different extensions and different pathways on the energy landscape. Both misfolding pathways originated from a partially folded on-pathway intermediate state, and they competed with native folding. The occupancy probability of both misfolded states could be controlled by modulating either the relaxation rate of the applied force and/or the calcium concentration. Remarkably for a calcium sensor, higher calcium concentrations, even within physiologically relevant conditions, lead to an increased probability of NCS-1 misfolding.