Compressive Force Activation of the Neuronal Nitric Oxide Synthase Enzyme

Abstract

Calmodulin (CaM), a calcium-sensing protein, regulates the production of nitric oxide (NO) by the nitric oxide synthase (NOS) enzyme. It is interesting to decipher the control mechanism of CaM-dependent NO biosynthesis by the NOS isoforms, as NO is a ubiquitous intercellular signaling molecule in numerous physiological processes. It is generally accepted that 4Ca²⁺-bound CaM associates with the NOS enzyme and activates NO production, while lower-level calcium has also been reported to allow for NOS enzymatic function under certain circumstances (e.g., shear stress). However, NO production by apo-CaM-activated NOS under an external force has not been directly demonstrated. Herein, we have utilized an atomic force microscopy (AFM)-correlated confocal microscopy technique to probe NO production by neuronal NOS (nNOS) enzyme, where compressive force-manipulated apo-CaM acts as a mechanosensing protein. DAR-4M was used as an NO-sensing fluorescent probe. Our results indicate that compressive force may induce necessary conformation changes of apo-CaM in binding and activating the nNOS enzyme.

Introduction

Nitric oxide synthase (NOS) is a redox enzyme catalyzing the production of nitric oxide (NO) from l-arginine (l-Arg). There are different isoforms of NOS found in various cell types, like neuronal, endothelial, glomerular, macrophage, renal tubular, and epithelial cells. − Three mammalian isoforms of NOS exist: neuronal, inducible, and endothelial NOS (nNOS, iNOS, and eNOS, respectively). nNOS is primarily expressed in both the central and peripheral nervous systems. It is also present in various nonneuronal cell types, including vascular smooth muscle cells, skeletal muscle fibers, the sarcoplasmic reticulum of cardiac myocytes, smooth muscle cells of the human coronary arteries, and intrinsic cardiac neurons from autonomic nerves and ganglia. − NO signaling in the human brain is involved in neuronal activity, such as learning and memory processes. In addition, nNOS-generated NO maintains vasodilation to some extent and helps in the regulation of microvascular tone, along with the regulation of cardiac rhythm and contractility by controlling parasympathetic and sympathetic nervous systems. ,, However, deviated NO production by nNOS has been implicated in pathologies of stroke and neurodegenerative diseases. There are several other important functions of eNOS-generated NO, such as maintaining cardiovascular homeostasis and inhibiting platelet aggregation. Hence, it is important to decipher the regulation mechanism of the NOS isoforms.

All three NOS isoforms are homodimers, and each subunit consists of an N-terminal heme-containing oxygenase domain and a C-terminal reductase domain. The terms of “heme domain” and the “oxygenase domain” are used interchangeably in the NOS field. This domain contains 6R-5,6,7,8-tetrahydrobiopterin (H₄B) and l-Arg-binding sites. The C-terminal reductase domain consists of NADPH-, FAD-, and FMN-binding sites. These two domains are connected by a linker region that binds calmodulin (CaM), a calcium-sensing protein (Figure ). CaM has four calcium-binding motifs. The binding of calcium to apo-CaM causes significant conformational changes, exposing hydrophobic amino acids that allow CaM to bind its target, such as the NOS enzyme. CaM binding activates the electron transfer from the reductase domain to the oxygenase domain, enabling NO production.

1.

Schematic representation of NOS domains and electron transport within the domains. (A) Intersubunit electron transport pathway in the active NOS. CaM binds to the linker region between the FMN and heme (oxygenase) domains. Electron transport across the NOS domains via the cofactors (FAD, FMN, and heme) is essential for producing NO at the catalytic heme site. The enzymatic reaction pathway, involving electron transfer and NO synthesis, has been represented by a reaction. Although the reaction proceeds through multiple steps, it is represented here with a single arrow for simplicity. (B) NOS domains organization showing two domains of a subunit.

CaM binds reversibly to nNOS and eNOS in response to increased Ca²⁺ concentrations. On the other hand, CaM binds tightly to the iNOS at basal [Ca²⁺]. Enzymatic reactions to synthesize NO by NOS require electron transport across the NOS domains (Figure A), where the FMN domain plays a key role. The FMN domain-mediated electron transfer is accompanied by the transfer of electrons to and out of the FMN cofactor. The FMN domain in NOS is highly dynamic, which allows it to gain alternative conformations among electron-accepting (docked FMN/FAD state) and electron-donating (docked FMN/heme state) conformations, as well as free, undocked conformations. All these pathways require CaM binding and association with NOS. − The majority of the literature reported the activation of nNOS and eNOS isoforms in a Ca²⁺-dependent manner. On the other hand, “Ca²⁺-independent” activation of eNOS has been observed, for example, in C2-ceramide-induced NO production and eNOS regulation by tyrosine phosphorylation of triton X-100 protein (cytoskeletal protein) under fluid shear stress. , Similarly, Hsp90 protein at a low concentration of calcium increases the affinity of CaM binding to eNOS, activating the enzyme at the basal calcium level. Additionally, atomic force microscopy (AFM) pulling experiments on the glypican-1 and heparan sulfate in the cells have reported the NOS activation and significant increase of NO production without added CaCl₂; AFM pulling has also been reported to induce the NO production through the cooperative mechanism of glypican-1 and PECAM-1. , We note that the so-called “Ca²⁺-independent” manner is not necessarily accurate, as basal-level calcium is still present; in fact, it is the EC₅₀ of calcium that can be lowered under these circumstances.

To our knowledge, low calcium level activation of the nNOS isoform has not been directly demonstrated, although there are a few studies done on the shear stress-induced activation of nNOS. For example, positive correlations have been observed between muscle activation and nNOS activity and NO concentration, particularly in response to mechanical loading in developing myotubes and skeletal muscle. However, important questions remain to be addressed, for example, whether stress within the cell affects nNOS activity and, if so, how? In this initial work, as part of a systematic study of the role of mechanical forces in the regulation of NOS activity, we have studied the activation of the nNOS enzyme by applying a compressive force by an AFM ultrasoft cantilever on apo-CaM, i.e., in the absence of calcium. AFM is a high-resolution technique that uses ultrasoft cantilevers to investigate the mechanical properties of biomolecules under physiologically relevant conditions. Biological molecules in cells are often subject to various mechanical forces, such as shear and compression. Thus, AFM serves as a highly advanced analytical tool for investigating cellular mechanics and structural features while also enabling real-time observation of enzymatic activity and protein behavior under externally applied mechanical perturbations at the piconewton (pN) level of force. −

Experimental Approach

Figure shows the combined AFM and confocal microscope setup for studying compressive force-induced apo-CaM activation of nNOS. We utilized an AFM to squeeze the apo-CaM molecules, where the CaM protein molecules come closer to each other. The external force provided by AFM builds up the stress within the CaM protein along with the protein molecules experiencing the force from one another due to the crowdedness. This mechanical force is inherent in a cellular environment, where biochemical signals are transduced by force-responsive proteins to maintain homeostasis and carry out cellular function. Inspired by the fact that NOS requires the association of CaM for NO synthesis, and given that CaM is presumably a force-responsive protein, , we aimed to investigate whether nNOS enzyme may produce NO in the presence of compressive force-activated CaM. In our force compression approach (Figure A–C), NO production by nNOS was fluorescently detected by diamino rhodamine-4M (DAR-4M; Figure E), a rhodamine-based dye used extensively for NO detection. NO reacts with DAR-4M in the presence of oxygen to give fluorescent diamino rhodamine-4M triazole (DAR-4M T; Figure F); DAR-4M T generated in the sample assay was excited at 532 nm, and the corresponding emission was recorded at 575 nm.

2.

Schematic representation of (A) combined AFM and confocal microscope setup in studying force-induced apo-CaM activation of the nNOS enzyme. (B) The inset shows the application of compressive force on apo-CaM immobilized on a cover glass using an AFM tipless cantilever; purple-colored particles are shown as nNOS enzyme in the sample mixture. Compressive force on apo-CaM alters the protein molecule conformation, increasing the binding affinity of CaM to nNOS and subsequent NO synthesis. (C) Covalently tethered CaM to the silane linker. (D) Apo-CaM bound state of nNOS, with the release of NO sensed by DAR-4M. NO reacting with DAR-4M gives fluorescent DAR-4M T. (E, F) Reaction shows the conversion of nonfluorescent DAR-4M into fluorescent DAR-4M T in the reaction assay.

Figure A illustrates how the compressive force-induced CaM-nNOS enzymatic reaction pathway may lead to NO release that can be detected by fluorescence of DAR-4M T; purple-colored molecules shown in Figure B are nNOS molecules in the assay medium. Figure B depicts a monolayer of CaM-tethered cover glass within the sample assay medium for force manipulation on CaM molecules, while Figure C shows a covalently linked CaM molecule to the silane linker. Figure D shows the detection of NO produced by nNOS in our force manipulation experiment (for experimental setup, see Section S1 in the Supporting Information).

Briefly, a compressive force was applied for 30 min using an AFM tipless cantilever in the approaching and retracting mechanism of the cantilever with a loading speed of 1000 nm/s. Here, the AFM cantilever (32 μm width × 350 μm length; see the SEM image in the Supporting Information, Section S2) acts as a flat ceiling under which a large number of apo-CaM molecules can be compressed and ruptured to yield different conformational states of CaM molecules. ,,, Since the size of the CaM protein is ∼5 nm, on average, there are about a few million CaM molecules that can be force-compressed and ruptured under a 32 μm × 32 μm area of the flat cantilever surface when the AFM cantilever contacts the cover glass for seconds. In seconds, millions of CaM molecules may undergo conformational change, promoting the ability to bind to the nNOS enzyme, which increases the enzymatic activity of nNOS. This cantilever compressive force application has millions of times more efficiency when compared to using an AFM tip with a ∼35 nm radius. In the 4Ca²⁺-CaM complex, CaM molecules will gain active conformations that have hydrophobic patches exposed in two lobes, allowing the association with its target proteins and peptides. On the other hand, under the compressive force, apo-CaM molecules can be ruptured, which will expose their binding sites for their target and gain binding activity comparable to that of 4Ca²⁺-CaM. It is found that the apo-CaM molecules undergo spontaneous rupture under compressive forces as low as 25–47 pN. Notably, the pN force in the living cell is biologically relevant due to the dynamic nature of the living cells that involves structural and thermal fluctuation under physiological conditions. −

Results and Discussion

We developed an AFM mechanical force based approach to manipulate the fluorogenic nNOS enzymatic reaction. A 50 μL sample assay containing all necessary substrates and cofactors was transferred to the cover glass tethered with a monolayer of apo-CaM. The assay medium consists of nNOS enzyme (700 nM), NADPH (100 μM), l-Arg (100 μM), H₄B (10 μM), as well as DAR-4M (200 nM), if added, in the assay buffer (50 mM Tris HCl, 10% glycerol (v/v), and 100 mM NaCl, pH 7.4). A CaM-tethered cover glass with 50 μL of assay medium was scanned over a 100 μm × 100 μm area with a confocal microscope (Axiovert 200, Zeiss) for 140 s to record the fluorescence intensity versus time during the initial phase of the reaction assay; in the initial phase of scanning, the reaction was initiated by adding nNOS and finally mixing DAR-4M in the assay; sample assay preparations and experimental data collection are described in the Supporting Information, Sections S6 and S7. DAR-4M dye readily reacts with NO to produce fluorescent DAR-4M T (Figure E,F). The quantum efficiency of DAR-4M T is the highest among DARs, and the detection limit of NO by DAR-4M is 5–10 nM, offering a good signal-to-noise ratio. This dye can detect NO above pH 4 and is quite stable in the assay solution. DAR-4M is highly sensitive and specific to NO radicals rather than reactive oxygen species (ROS) and reactive nitrogen species (RNS), even if they are generated in very low concentrations in the reaction assay. Additionally, DAR-4M has poor-to-no direct reactivity to RNS. , To minimize the generation of ROS and RNS, the enzymatic NOS assay was conducted under carefully controlled conditions. Fresh and sufficient amounts of H₄B and l-Arg were supplied to minimize nNOS uncoupling and the subsequent electron transfer to molecular oxygen, which leads to superoxide formation. The assay employed buffers free of metal ions, phosphates, and oxidizing agents and was maintained at an optimal pH of 7.4. Additionally, NADPH was used at concentrations that ensured a proper electron flow within the catalytic cycle. Hence, in our force manipulation assay, it is unlikely that the ROS or RNS can be generated by force manipulation. Furthermore, even if generated in a very low concentration, the potential impact of the ROS and RNS on the DAR-4M dye fluorescence is minimal, as shown in Figure by a comparison of experiments I, II, and III vs IV and V, where signals of all experiments are expected to be impacted by RNS and ROS to the same extent if there’s any. A significant difference in the fluorescence signal was observed when comparing control experiments I, II, and III with IV and V, indicating NOS activation and subsequent NO production, independent of potential RNS or ROS involvement in NO detection by the fluorescence dye.

5.

Scatter plot distribution of mean intensity percentage change for 10 sets of replicates in compressive force experiments (I–IV), along with mean intensity percentage change of initial and final scans in the no-force experiment (V). Central solid lines represent the mean of the mean intensity percentage change, and the rings in the figure represent the mean intensity percentage change for 10 replicates.

Trace (I) in Figure A shows a section (20 s) of the recorded fluorescence trajectory before applying force. We observed some background fluorescence in this initial scan, which can be due to the autofluorescence of DAR-4M dye in the absence of NO. To achieve a good fluorescence signal and minimize autofluorescence from the dye, we reduced the concentration of dye to 200 nM. , After the initial photon counts recording, the laser and AFM cantilever were coaxially aligned (see Section S3 in Supporting Information). Finally, after the alignment, we compressed the tethered apo-CaM molecule continuously by approaching and retracting the cantilever. We tethered the CaM molecules on the cover glass but not the nNOS since we did not observe any ruptured events of the nNOS enzyme under the compressive force by AFM (see Section S4 in the Supporting Information). Notably, CaM molecules spontaneously ruptured under the compressive force of the AFM tip (see Section S4 in the Supporting Information). The property of the protein to show the rupture events under compression can be a protein-specific behavior. This behavior depends on the structural rigidity or flexibility of the globular protein, where each domain in the protein may behave differently. Some protein behaves like a balloon, which can hold some compressive stress and show sudden rupture behavior under compression, whereas some proteins may behave as a cotton ball-like structure, which does not hold any compressive stress and does not show rupture events. , A Cr–Au-coated AFM tipless cantilever (MicroMash) of 32 μm × 350 μm (width × length) was used to apply external compression to the CaM proteins. As soon as the cantilever comes in contact with the sample surface, the CaM molecules experience continuous loading of force due to the bending of the cantilever as a result of piezo displacement. The piezo displacement and cantilever bending continue for 1 s, applying compressive force in a few nanonewtons (nN) (see Section S5 in the Supporting Information). The compressive force was applied for 30 min with a series of force loading repetition rates with constant force loading velocity (1000 nm/s) to rupture the apo-CaM and to facilitate conformational change induced by mechanical stress. Here, the repetition rate defines the total number of force–distance spectroscopy cycles within 30 min at one point on the monolayer CaM-modified cover glass.

3.

General workflow of fluorescence trajectory data collection and analysis. (A) Fluorescence intensity vs part of time trajectory (20 s) recorded before (I) and after (II) applying compressive force; (B, D) distribution of the fluorescence intensity over a 140 s time course before (I) and after (II) applying pN compressive force. The bar graph in (C) represents the mean fluorescence intensity before and after force application in calculating the percentage of intensity increase due to mechanical stress on protein molecules.

We found that the 30 min force application was sufficient for the simultaneous rupture of protein and promoting the association of CaM with nNOS. Force manipulation was carried out under complete dark conditions, and the sample in the same force-compressed area was then illuminated by the excitation laser to observe the fluorescence (trace II in Figure A). After 30 min of continuous force application in the approach and retract cycle, the fluorescence intensity versus time trajectory was recorded for 140 s at the same area where the force was applied (trace II in Figure A). To analyze the raw fluorescence data versus time, we plotted the histograms of intensity versus time trajectory to calculate the mean fluorescence intensity before and after the applied force (Figure B,D). In our analysis, we calculated the percentage change of the mean intensity before and after applying force. The percentage change of mean intensity gives us the “specific” fluorescence intensity due to the formation of DAR-4M T. The bar graph in Figure C shows the mean fluorescence intensity before and after applying force. We used the general data collection and analysis workflow for the experiments below.

Our compressive force experiments consist of 4 sets of data: 3 control experiments (I–III in Figure ) and 1 experiment of detection of NO produced by nNOS (IV in Figure ), all experiments under compressive force, carried out at the force loading repetition in a 6 s time interval. We also did a no-force experiment (V in Figure ) with added calcium chloride to activate nNOS, where fluorescence intensity was recorded for the initial scan and final scan after waiting for 30 min, but without applying force. We carried out 10 replicates for each set of experiments.

4.

Average of mean fluorescence intensity before and after force application in the compressive force experiments (I–IV), along with average of mean fluorescence intensity of initial and final scans in no-force experiments (V). The experimental conditions are described in the bar graph.

Figure shows the average of mean fluorescence intensity before and after force from 10 replicates for the four sets of experimental data from the compressive force experiments that include controls (samples I, II, and III) and the NO synthesis by apo-CaM-bound nNOS (sample IV). All of these samples did not contain added calcium in the assay medium. Samples I and II did not contain the nNOS enzyme or NADPH substrate, respectively, and could thus be used as negative controls. Sample III is another negative control (IV), where the cover glass was tethered with Replication Protein A (RPA-14 protein), a protein of comparable size to CaM; RPA-14 does not bind to nNOS, though. Figure (IV) shows the average of the mean fluorescence intensity before and after the compressive force calculated from 10 replicates of the experiments, and this experiment represents the activation of nNOS by apo-CaM under the compressive force. Of note is that sample IV contains all the necessary reagents and proteins required for NO production, except calcium ions. As a positive control (V), nNOS was activated in the presence of 4Ca²⁺-bound CaM without compressive force. In this no-force experiment, we did an initial confocal scan in 100 μm × 100 μm for a 50 μL assay medium and waited for 30 min instead of applying force. A final confocal scan was then done in the same area to record the fluorescence trajectory over 140 s. We then calculated the percentage change from the mean intensity in each trial.

Figure shows the mean intensity percentage change for each set of experiments. The mean intensity percentage change was calculated using the ratio of (mean intensity after the force–mean intensity before the force)/mean intensity recorded before the force. The mean intensity percentage change is in the range of 20–60% for samples I, II, and III, i.e., the negative controls. Importantly, the mean intensity percentage change is in the range of 50–200% for samples IV and V. Specifically, the mean of the mean intensity percentage change for sample IV is 100%, which is at least 2 times that of the controls (20–60%). Moreover, the Ca²⁺-repleted experiment (sample V) gave a similar percentage intensity change as sample IV (see Section S8 in the Supporting Information for the graph and percentage Table S1). In other words, compression-force-activated CaM and 4Ca²⁺-bound CaM stimulate the nNOS enzyme to a similar extent. We note that the trials in cases IV and V have shown significantly higher fluorescence percentage increases compared to trials I, II, and III, although the increases are only about a factor of 2; these increases are statistically significant beyond two standard deviations. Furthermore, both assay results from the force-CaM activated enzymatic reaction versus the Ca²⁺-CaM activated enzymatic reaction show comparable NO productivity within the error bars. The error bars may arise from the inhomogeneous and complex mechanism of protein–protein, enzyme–substrate, and enzyme–protein interactions. For example, protein interaction dynamics can vary from molecule to molecule, site to site, and time to time for the same individual molecule due to the spatial and temporal inhomogeneity of protein molecules in the solution, thereby affecting the enzymatic reaction turnover and product-releasing tendency of enzymes. , To confirm that the nNOS enzyme was active, we conducted a hemoglobin-NO capture assay (see Section S11 in the Supporting Information). The NO production activity is 54.2 ± 0.5 min^–1, which is consistent with the literature value; as expected, no activity was observed in the absence of CaM in such assay.

We recognize that the reactivity and sensitivity of any fluorescent probe may be affected by the components in the assay medium, including the size of the added proteins and the different amino acid residues present in the protein molecule due to the change in hydrophobicity. We thus performed compression force experiments for nNOS in the presence of RPA14, a protein of comparable size that does not bind to NOS. RPA-14 was tethered on the cover glass surface via a silane-based linker following a protocol analogous to that used for CaM. The mean fluorescence intensity percentage change upon force application was similar to that of samples I and II, where NO should not be produced due to the absence of the NOS enzyme or the NADPH substrate.

These results collectively demonstrate that it is the production of NO by the nNOS enzyme that is responsible for the observed significant increase in mean fluorescence intensity upon application of a compression force to apo-CaM in sample IV. We observed a broad range of percentage change for samples IV and V (Figure ), where the lower regions somewhat overlapped with the higher regions of the control samples. The broad range of percentage change indicates the inhomogeneous and complex nature of the protein and enzyme in the local environment in the experiment, which involves the different orientations of the molecules on the surface and in the sample assay, and the inhomogeneity of electrostatic, hydrophilic, and hydrophobic force fields around the molecule under compression. Some of the tethered CaM molecules may not allow for sufficient conformational space to support additional CaM docking to the NOS domains and higher-order domain rearrangement required for efficient ET across the NOS domains. , That being said, some of the runs did show more than two times the mean intensity percentage change when compared to our negative controls, and the mean changes are indeed statistically much higher than the control groups (Figure ). Hence, our experimental data demonstrate that there is a significant increase in the NO production activity of nNOS by applying compression force alone on apo-CaM. Our experimental results demonstrate, for the first time, that nNOS can be activated by CaM via compression forces in the absence of added calcium ions.

The cellular milieu experiences the force of different rates during blood flow, muscle contraction and relaxation, neuronal activity, glial cell function, and the interplay between various types of cells. Hence, to study the effect of force at various repetition rates as an additional control, we performed AFM force loading manipulations at different repetition rates to detect nNOS activation and examine the effect of increasing the force loading repetition rate on NO production. As shown in Figure , we repetitively applied force for 30 min at different time intervals. We increased the force loading repetition rate by decreasing the time interval between each cycle of the AFM tip approach to and retraction from the sample surface. The average of mean intensity percentage changes of 5 replicates at 5 min force loading intervals was found to be 68%, whereas at 2 min intervals, it was 82%, and at 30-s intervals, it was 85%, where mean intensity percentage change was calculated using the ratio of (mean intensity after the force–mean intensity before the force)/(mean intensity recorded before the force) (see Section S9 in the Supporting Information for graph and percentage; Table S2). Our results suggest that when the force applied became more frequent, specifically with a 30 s interval, it led to the enhancement of the nNOS accumulated productivity of NO. Hence, this control also supports the activation of nNOS under compressive force manipulation, as we noticed the increase in the mean intensity percentage change upon increasing the repetition of the applied force. Data in Figure IV were achieved with the highest repetition of 6 s time intervals. Intriguingly, elevated activity of nNOS in response to increased mechanical force may be associated with enhanced mechanical stress resulting from stretching or compression within the cellular environment.

6.

Scatter plot distribution of frequency-dependent mean intensity percentage change for five sets of replicates in the compressive force experiment. The experimental conditions are the same as in sample IV in Figure .

Although no Ca²⁺ was added in our compression force experiments and nanopure deionized water was used throughout, it cannot be completely ruled out that a residual amount of Ca²⁺ might still be present and bind to the CaM protein (see Section S10 in the Supporting Information). However, such residual Ca²⁺ concentrations are insufficient to make 4Ca²⁺-bound CaM, the predominant form. This supports our observation that nNOS was activated only by repetitive compressive force manipulations (Figures IV and ).

Figure shows a conceptual model of the CaM-nNOS complex under a mechanical compression force. The canonical CaM-binding site in NOS is situated within the linker connecting the FMN and heme domains, and additional interprotein docking interactions between the CaM and the NOS domains further facilitate orienting the oxygenase and reductase domains for efficient electron transport, making CaM an essential molecular switch of NOS. The association and interplay between CaM and NOS play key roles in changing the conformation of NOS and allowing electron transport across the NOS domains. Our research herein demonstrates that this association between CaM and NOS can also be facilitated by mechanical force in vitro and likely inside the cell. Additionally, CaM functions as a molecular bridge, facilitating interdomain communication by mediating interactions between the oxygenase and reductase domains of NOS. This bridging extends beyond the linker region, with the FMN subdomain of the reductase also contributing to interactions with the oxygenase domain. Additionally, the interaction between CaM and nNOS is reversible and associated with noncovalent interactions, hydrogen bonds, and hydrophobic interactions. ,− Based on all these facts, our experimental data have clearly shown that compressive force on CaM can activate nNOS via various complex mechanisms of CaM–nNOS interaction.

7.

Conceptual model showing two activation pathways of nNOS by CaM: calcium-sensitive pathway (I) vs mechanosensitive pathway (II). Apo-CaM (A), upon applying compression force (C), leads to force-activated CaM molecules in different conformations (D). On the other hand, calcium (black dots in B)-bound CaM exhibits similar conformations where the CaM central linker region is exposed to interact with the target NOS protein. The CaM states in B and D putatively bind to and activate nNOS (light orange colored) in E. Only one NOS subunit is shown here for clarity.

Every cell in the body is exposed to mechanical force and responds to this force to some extent. There are several externally applied forces to the cell in biology, such as shear stress, compression, tension, and cell-generated forces, which affect the structural behavior of the protein molecules and biological functions of the living entity. − Mechanical force developed inside the cell tends to regulate the activity of proteins by rearranging the protein domains and conformations. There is a dynamic relationship between protein conformation and its biological functions in the body, which has been extensively studied. − It has been previously reported that CaM, in addition to its role as a calcium-sensitive protein, is a mechanosensitive protein and can undergo spontaneous rupture under ∼35 pN force, a biologically relevant force amplitude in the living cell. Although the mechanism of how ruptured CaM binds to and activates nNOS is unclear, our initial results in the present work demonstrate that these compressive force-ruptured CaM proteins can activate the nNOS enzyme in catalyzing the production of NO. Our compressed force experimental analysis indicates that nNOS can still be activated in the absence of increased calcium concentrations, and even a minimal production of NO should not be overlooked, as it may be biologically relevant due to mechanical stress on CaM.

The mechanistic interaction of CaM with NOS and the exact conformation of the CaM-NOS complex are unclear and yet to be explored. Nonetheless, experimental studies have shown that CaM undergoes force-induced conformational changes under conditions of macromolecular crowding, thereby facilitating its activation for interaction with target molecules. Similarly, AFM studies have demonstrated that CaM can adopt a conformation capable of binding the CW28 peptide when subject to abrupt and spontaneous rupture under piconewton-range compressive force. The study of different conformational states of CaM achieved under compressive force and the mechanism of nNOS–CaM interaction is beyond the scope of this study.

In recent years, studies have reported the activation of NOSs in different signaling pathways that increased the production of NO. Fluid shear stress can enhance the NO formation via Ca²⁺-independent tyrosine kinase inhibitor-sensitive pathways. Moreover, in fluid shear stress, several kinase inhibitors have been found to enhance the endothelium-derived NO production without an increase in the intracellular calcium ions, such as shear stress can activate a signaling pathway that involves inhibition of phosphoinositide 3-Kinase (PI3K) and phosphorylation of the serine/threonine residue in eNOS. Mechanical perturbation caused NO production by sensitizing RBC-eNOS. Phosphorylated eNOS activity was increased at resting [Ca²⁺]. Red blood cells (RBCs) suspension exposure to the shear stress increased the NO level. Apart from shear stress-induced activation, some studies have reported that isometric contraction and isometric stretch can activate eNOS via a Ca²⁺-independent, tyrosine kinase inhibitor-sensitive pathway. Additionally, flow-induced NOS activation mediated by protein kinase N2 (PKN2) has been reported. , The living cell consists of a large number of macromolecules that can affect the conformational dynamics of the protein molecules inside the cell. For example, in an enzymatic reaction, the effect of macromolecular crowding on iNOS activity showed an increase in the interdomain electron transport. ,, Although immense results have shown the enhanced production of NO in a number of different pathways, it remains unexplored and debatable how exactly the NO production activity of NOS is regulated by these extrinsic cues.

Living cells are dynamic systems that involve structural and thermal fluctuations under physiological conditions. These fluctuations create a transient and temporarily anisotropic force field, which can trigger many biological processes related to force activation and modify enzyme kinetics and activity at the molecular level. For example, different proteins and enzymes are subject to various forces inside the cells. Our experimental condition mimics such an anisotropic force vector and helps study the force-modulated activation of nNOS. Our experiment indicates that the mechanical force-driven mechanism can lead to an increase in NO production, alongside the other biochemical pathways, including the Ca²⁺/CaM complexation pathway.

Conclusions

It is generally accepted that the activation of nNOS and eNOS isoforms is associated with the binding of the Ca²⁺/CaM complex to NOS, triggered by an increase in the Ca²⁺ concentration. Here, we observed that the nNOS enzyme can be activated by promoting force-induced binding of CaM to nNOS under a compressive force from the AFM cantilever, even in the absence of added Ca²⁺. The CaM protein conformational rupture occurs spontaneously under pN compressive force. The subsequent conformational changes of CaM putatively support the binding of CaM to nNOS, even without added calcium. Our result suggests that, beyond the Ca²⁺ chemical activation pathway, force activation may also play a significant role in the protein function dynamics under conditions in which calcium concentration does not increase, thereby influencing nNOS enzymatic activity on NO production in living cells.

Glossary

Abbreviations

NO

nitric oxide

NOS

nitric oxide synthase

nNOS

neuronal NOS

CaM

calmodulin

H₄B

6R-5,6,7,8-tetrahydrobiopterinn

l-Arg

l-arginine

AFM

atomic force microscopy

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c03891.

• Experimental setup used in our analysis (Figure S1); SEM image of AFM probe (Figure S2); procedure of coaxial alignment of laser (Figure S3); force curve due to compressive force manipulation (Figure S4); typical force curve (Figure S5); chemical modification of cover glass (Figure S6); procedure involved in our experiment (Figure S7); averages of mean intensity % change (Figure S8); AFM force loading repetition rate-dependent mean of mean intensity % change (Figure S9); average of mean intensity % change values (Tables S1 and S2); and native protein mass spectra of CaM protein (Figure S10) (PDF)

H.P.L. and C.F. designed the research and provided the analytical tools. L.S., Y.P.G., T.J., D.S.S. performed research; L.S., Y.P.G., T.J., D.S.S., C.F., H.P.L. analyzed data and wrote the paper. All authors have approved the final version of the manuscript.

H.P.L. acknowledges the support from the Ohio Eminent Scholar Endowment. C.F. acknowledges support from the National Institutes of Health GM156443 and the National Science Foundation 2041692. This work was partly supported by the National Institutes of Health P20GM130422 and P30ES032755.

The authors declare no competing financial interest.