Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics Yufan Hea,1, Mohammad Mahfuzul Haqueb,1, Dennis J. Stuehrb,2, and H. Peter Lua,2 aCenter for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403; and bDepartment of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 Edited by Louis J. Ignarro, University of California, Los Angeles School of Medicine, Beverly Hills, CA, and approved July 31, 2015 (received for review May 5, 2015) Mechanisms that regulate the nitric oxide synthase enzymes (NOS) this model, the FMN domain is suggested to be highly dynamic are of interest in biology and medicine. Although NOS catalysis and flexible due to a connecting hinge that allows it to alternate relies on domain motions, and is activated by calmodulin binding, between its electron-accepting (FAD→FMN) or closed confor- the relationships are unclear. We used single-molecule fluores- mation and electron-donating (FMN→heme) or open conforma- cence resonance energy transfer (FRET) spectroscopy to elucidate tion (Fig. 1 A and B)(28,30–36). In the electron-accepting closed the conformational states distribution and associated conforma- conformation, the FMN domain interacts with the NADPH/FAD tional fluctuation dynamics of the two electron transfer domains domain (FNR domain) to receive electrons, whereas in the elec- in a FRET dye-labeled neuronal NOS reductase domain, and to tron donating open conformation the FMN domain has moved understand how calmodulin affects the dynamics to regulate away to expose the bound FMN cofactor so that it may transfer catalysis. We found that calmodulin alters NOS conformational electrons to a protein acceptor like the NOS oxygenase domain, or behaviors in several ways: It changes the distance distribution to a generic protein acceptor like cytochrome c. In this way, the between the NOS domains, shortens the lifetimes of the individual reductase domain structure cycles between closed and open con- conformational states, and instills conformational discipline by formations to deliver electrons, according to a conformational greatly narrowing the distributions of the conformational states equilibrium that determines the movements and thus the electron and fluctuation rates. This information was specifically obtainable flux capacity of the FMN domain (25, 28, 32, 34, 35, 37). A similar only by single-molecule spectroscopic measurements, and reveals conformational switching mechanism is thought to enable electron how calmodulin promotes catalysis by shaping the physical and transfer through the FMN domain in the related flavoproteins temporal conformational behaviors of NOS. NADPH-cytochrome P450 reductase and methionine synthase reductase (38–42). conformational motion | flavoprotein | domain–domain interaction | NOS enzymes also contain a calmodulin (CaM) binding domain FRET | electron transfer that is located just before the N terminus of the FMN domain (Fig. 1B), and this provides an important layer of regulation (25, 27). lthough proteins adopt structures determined by their amino CaM binding to NOS enzymes increases electron transfer from Aacid sequences, they are not static objects and fluctuate NADPH through the reductase domain and also triggers electron among ensembles of conformations (1). Transitions between transfer from the FMN domain to the NOS heme as is required for these states can occur on a variety of length scales (Å to nm) and NO synthesis (31, 32). The ability of CaM, or similar signaling time scales (ps to s) and have been linked to functionally relevant proteins, to regulate electron transfer reactions in enzymes is un- phenomena such as allosteric signaling, enzyme catalysis, and usual, and the mechanism is a topic of interest and intensive study. protein–protein interactions (2–4). Indeed, protein conforma- It has long been known that CaM binding alters NOSr structure tional fluctuations and dynamics, often associated with static and such that, on average, it populates a more open conformation (43, dynamic inhomogeneity, are thought to play a crucial role in 44). Recent equilibrium studies have detected a buildup of between biomolecular functions (5–11). It is difficult to characterize such spatially and temporally inhomogeneous dynamics in bulk solu- Significance tion by an ensemble-averaged measurement, especially in pro- teins that undergo multiple-conformation transformations. In Electron transfer is a fundamental process in biology that can such cases, single-molecule spectroscopy is a powerful approach be coupled to catalysis within redox enzymes through a careful to analyze protein conformational states and dynamics under phys- control of protein conformational movements. Using single- iological conditions, and can provide a molecular-level perspective molecule fluorescence resonance energy transfer (FRET) spec- on how a protein’s structural dynamics link to its functional – troscopy, we find that calmodulin binding to neuronal NO mechanisms (12 21). synthase reductase domain (nNOSr) both alters and restricts A case in point is the nitric oxide synthase (NOS) enzymes – the distributions of NOSr conformational states and the con- (22 24), whose nitric oxide (NO) biosynthesis involves electron formational lifetimes, thereby revealing a physical means by transfer reactions that are associated with relatively large-scale which calmodulin may control electron transfer for catalysis. A movement (tens of Å) of the enzyme domains (Fig. 1 ). During BIOCHEMISTRY catalysis, NADPH-derived electrons first transfer into an FAD Author contributions: M.M.H., D.J.S., and H.P.L. designed research; Y.H. and M.M.H. per- domain and an FMN domain in NOS that together comprise the formed research; Y.H., M.M.H., D.J.S., and H.P.L. contributed new reagents/analytic tools; NOS reductase domain (NOSr), and then transfer from the Y.H. and M.M.H. analyzed data; and Y.H., M.M.H., D.J.S., and H.P.L. wrote the paper. FMN domain to a heme group that is bound in a separate at- The authors declare no conflict of interest. tached “oxygenase” domain, which then enables NO synthesis to This article is a PNAS Direct Submission. begin (22, 25–27). The electron transfers into and out of the 1Y.H. and M.M.H. contributed equally to this work. FMN domain are the key steps for catalysis, and they appear to 2To whom correspondence may be addressed. Email: [email protected] or [email protected]. rely on the FMN domain cycling between electron-accepting and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. electron-donating conformational states (28, 29) (Fig. 1B). In 1073/pnas.1508829112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1508829112 PNAS | September 22, 2015 | vol. 112 | no. 38 | 11835–11840 Downloaded by guest on September 29, 2021 domain interface, and (ii) expected to fluctuate within a distance range of ∼40–60 Å for the closed and maximally open states, respectively (Fig. 1 A and B), which allows for monitoring of conformational changes by FRET of the Cy3–Cy5 pair, using a corrected Forster radius of Ro, 45 Å that we determined for the dyes when bound to the nNOSr protein. The E827C/Q1268C variant could be labeled with the Cy3 and Cy5 maleimide dyes to near stoichiometry (1.1 and 0.9 mol dye per mol enzyme, respec- tively) (Fig. S1), and the Cy3–Cy5-labeled enzyme displayed a normal catalytic activity and CaM response compared with native nNOSr enzyme or to the CLnNOSr precursor enzyme (Fig. 1C). For the FRET spectroscopic experiments, single molecules of dye- labeled nNOSr were entrapped in chambers formed within a buff- ered 1% agarose gel, then the single-molecule FRET fluctuation time trajectories were recorded under a home-modified single-mol- ecule spectroscopic imaging microscope. A detailed description of Fig. 1. (A) The nNOSr ribbon structure (from PDB: 1TLL) showing bound the experimental set up and approaches has been published (14, 15). FAD (yellow) in FNR domain (green), FMN (orange) in FMN domain (yellow), A B – – connecting hinge (blue), and the Cy3–Cy5 label positions (pink) and distance Fig. 2 and shows typical donor acceptor (D A) two-channel (42 Å, dashed line). (B) Cartoon of an equilibrium between the FMN-closed fluorescence images collected for single molecules of Cy3–Cy5 la- and FMN-open states, with Cy dye label positions indicated. (C) Cytochrome beled nNOSr within a 10 μm×10 μm laser confocal scanning area in c reductase activity of nNOSr proteins in their CaM-bound and CaM-free the agarose gel. Each feature in the images is attributed to a single states. Color scheme of bar graphs: Black, WT nNOSr unlabeled; Red, Cys-lite nNOSr molecule; the intensity variation between the molecules is (CL) nNOSr unlabeled; Blue, E827C/Q1268C CL nNOSr unlabeled; and Dark due to FRET and the different longitudinal positions in the light cyan, E827C/Q1268C CL nNOSr labeled. focal depths. Protein conformational changes that cause changes in FRET D–A distance result in coincident and opposite, anti- correlated donor and acceptor fluorescence signal fluctuations two to four discreet conformational populations in NOS enzymes (donor decreases, acceptor increases, and vice versa). Fig. 2C and in related flavoproteins, and in some cases, have also estimated shows the intensity vs. time trajectories of the Cy3 donor [Id(t), the distances between the bound FAD and FMN cofactors in the green]- and Cy5 acceptor [Ia(t), red]-labeled nNOSr under the different species (26, 36, 37, 39, 40), and furthermore, have con- CaM-free condition. Typically, single-molecule D–A signal fluctu- firmed that CaM shifts the NOS population distribution toward ation involves not only FRET-related fluctuation, which shows the more open conformations (34, 36, 45). Although valuable, such anti-correlated relation between the D–A signal fluctuations; but ensemble-averaged results about conformational states cannot ex- also noncorrelated thermal-related random fluctuation, and mea- plain how electrons transfer through these enzymes, or how CaM surement noise.