Role of Dimers in the cAMP-Dependent Activation of

Jan 11, 2018 - The resulting allosteric model provides a viable rationalization of electrophysiological data on the role of IR dimers in. HCN activati...
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Role of Dimers in the cAMP-Dependent Activation of Hyperpolarization-Activated Cyclic-Nucleotide-Modulated (HCN) Ion Channels Bryan VanSchouwen† and Giuseppe Melacini*,†,‡ †

Department of Chemistry and Chemical Biology and ‡Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada S Supporting Information *

ABSTRACT: Hyperpolarization-activated cyclic-nucleotidemodulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity through the cAMP-induced formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN. Although evidence from confocal patch-clamp fluorometry indicates that the cAMP-dependent gating of HCN occurs through a dimer of dimers, the structural and dynamical basis of cAMP allostery in HCN dimers has so far remained elusive. Thus, here we examine how dimers influence IR structural dynamics, and the role that such structural dynamics play in HCN allostery. To this end, we performed molecular dynamics (MD) simulations of HCN4 IR dimers in their fully apo, fully holo, and partially cAMP-bound states, resulting in a total simulated time of 1.2 μs. Comparative analyses of these MD trajectories, as well as previous monomer and tetramer simulations utilized as benchmarks for comparison, reveal that dimers markedly sensitize the HCN IR to cAMP-modulated allostery. Our results indicate that dimerization fine-tunes the IR dynamics to enhance, relative to both monomers and tetramers, the allosteric intra- and interprotomer coupling between the cAMP-binding domain and tetramerization domain components of the IR. The resulting allosteric model provides a viable rationalization of electrophysiological data on the role of IR dimers in HCN activation.



INTRODUCTION Hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels are cyclic AMP (cAMP)-regulated proteins that play a key role in nerve impulse transmission and heart rate modulation in neuronal and cardiac cells, respectively.1−10 All HCN isoforms contain an N-terminal transmembrane (TM) region, which forms a tetrameric assembly harboring the ion pore, and a C-terminal intracellular region (IR) that confers regulation by cAMP (Figure 1a,b).1,2,5,7,9−13 The transmembrane (TM) and intracellular (IR) regions of HCN are highly conserved at the sequence level among vertebrates, with sequence conservation of 91−97% and sequence identities of 80−93%.14 It has been suggested that tetramerization of the HCN IR in response to cAMP binding is closely linked to the cAMPdependent upregulation of ion-channel opening.3,15−17 Specifically, the HCN IR tetramer adopts an “elbow−shoulder” topology3 involving intermonomer interactions of two α-helical hairpins (αA′−αB′ and αC′−αD′) at the IR N-terminus, which are referred to as the “tetramerization domain” (TD; Figure 1a,b). Meanwhile, cAMP-binding domains (CBDs) are located C-terminal to the TDs of each monomer and include αE′, αF′ and αA helices that form the so-called N3A motif, as well as a central β-subdomain and C-terminal αB and αC helices (Figures 1a,b and S1a). These CBDs allosterically control TD © XXXX American Chemical Society

self-association, whereby cAMP binding to the CBD promotes TD tetramerization.3 The cAMP-dependent tetramerization is expected to be particularly relevant for the HCN isoforms abundant in brain and heart tissues (i.e., HCN2 and HCN4), but less so for other HCN isoforms such as HCN1. Indeed, structures of intact HCN1 from cryo-electron microscopy18 show that the HCN1 IR already exists as a tetramer in the apo state, and it has been previously demonstrated that the apo-state IR of HCN1 has a significantly greater propensity for tetramerization than the apo-state IR of either of the other two cAMP-responsive isoforms (i.e., HCN2 and HCN4).17 These observations suggest that although HCN1 experiences a reduced degree of perturbation by cAMP,17 cAMP-dependent tetramerization cannot be overlooked for HCN2 and HCN4 and thus deserves further scrutiny. Previously, Akimoto et al. (2014)19 solved the inactive apostate structure of the HCN4 CBD (Figure S1b) and found that it differs from the active cAMP-bound structure (Figure S1a)5 by a rearrangement of the α-helical CBD components similar to that observed for the CBDs of other cAMP receptors such as Received: October 12, 2017 Revised: January 11, 2018

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Figure 1. Introduction and design of the comparative simulation analyses. (a, b) Ribbon-structure representation of (a) the active tetrameric structure of the HCN4 IR bound to four molecules of cAMP (PDB ID “3OTF”), and (b) the cAMP-bound dimeric structure derived from it. The four IR protomers are shown in red, green, blue and orange, and key protomer structural elements are labeled in (b): the αA′−αD′ helices that form the “tetramerization domain” (TD) of the IR; and the “N3A” motif (αE′−αF′−αA), central β-subdomain (“β-core”), and C-terminal helices (αB−αC) of the cAMP-binding domain (CBD). Bound cAMP molecules are shown as blue sticks, and for clarity, the boundary between the TD and CBD regions is indicated by black dashed lines. Schematic representations of the tetramer and dimer are shown below the ribbon structures, with protomers 1 and 2 (“P1” and “P2”) indicated for the dimer, and bound cAMP illustrated as filled blue squares. (c) Schematic outline of the four dimer structures derived from the cAMP-bound dimer shown in (b), and the monomers, dimers and tetramers with which each of the four dimers is compared to assess how the dimers influence IR structural dynamics. The figures in which the comparisons are analyzed are indicated, along with a brief explanation of the attribute(s) of dimer structural dynamics assessed by each comparison.

PKG, PKA, and EPAC (Figure S1c).20−39 Similar apo-state overall CBD structures were subsequently reported for HCN2, highlighting the relevance of such CBD structural rearrangement for allostery of other HCN isoforms.40,41 On the basis of these structures, it was proposed that the inactive CBD conformation destabilizes the tetramer through steric clashes with the TD, which arise as the result of an incompatibility of the inactive CBD conformation with the tight steric packing imposed by tetramer assembly.19 Stabilization of an active CBD conformation upon cAMP binding removes these steric clashes, thus providing an explanation for how cAMP controls HCN channel gating via TD tetramerization.19,42 Alternatively, in the absence of cAMP, the CBD/TD steric clashes can be removed

through dissociation of the tetramer into monomers, which results in increased TD flexibility and provides an explanation for the ion-channel autoinhibition imposed by the apo-state CBD.42 Although this model19,42 provided a viable explanation for cAMP-dependent modulation of HCN IR tetramerization, it had also been hypothesized that the CBD/TD steric clashes could potentially be removed through dissociation of the tetramer into dimers rather than monomers.19 Indeed, subsequent molecular dynamics (MD) simulations starting from “hybrid” HCN4 IR tetramer structures highlighted two alternative pathways through which the CBD/TD steric clashes can be removed.43 Specifically, it was found that lowering the B

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Figure 2. (a, c, e, g) Distributions of backbone root-mean-square deviations (RMSDs) of the full IR and the tetramerization domain (TD) from their configurations in the initial 3OTF structure, as observed in the context of the apo/monomer (gray),42 protomer 1 of the fully apo dimer (red), protomer 2 of the fully apo dimer (green), and the apo/tetramer (black)42 of the HCN4 IR (schematically outlined in the left inset panel). (a) RMSDs for the entire HCN4 IR, (c) RMSDs for the TD, (e) RMSDs within the αA′−αB′ segment of the TD, (g) RMSDs within the αC′−αD′ segment of the TD. (b, d, f, h) As in (a, c, e, g), but for the respective holo states (i.e., holo/monomer, fully holo dimer, and holo/tetramer; schematically outlined in the right inset panel). All boxplots were constructed on the basis of the RMSD data from all replicates for each state. The statistics reported in each boxplot are as follows: the middle, bottom, and top lines of the central box represent the median, 25th percentile, and 75th percentile of the data set, respectively; the whiskers represent additional data falling within 1.5 × IQR above the 75th percentile or below the 25th percentile (where the interquartile range, or IQR, is the difference between the 75th and 25th percentiles); the “■” symbol represents the mean of the data set; and the “▲” and “▼” symbols represent the 1st and 99th percentiles of the data set, respectively. C

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Figure 3. (a) Backbone root-mean-square fluctuations (RMSFs) vs residue number plots for the apo/monomer (brown),42 protomer 1 of the fully apo dimer (red), protomer 2 of the fully apo dimer (green), and the apo/tetramer (black)42 of the HCN4 IR (schematically outlined in the top inset), averaged across all replicates for the respective states. (b) As in (a), but for the respective holo states (i.e., holo/monomer, fully holo dimer, and holo/tetramer; schematically outlined in the bottom inset). The secondary structure elements are indicated along the horizontal dimensions of both graphs (black bars = α-helices, brown bars = β-strands), and structural regions exhibiting notable differences among states are indicated (dotted rectangles).

undergo inactive-to-active transitions in a highly cooperative manner. Therefore, in the current work, we sought to more closely examine how dimers influence IR structural dynamics, and the role that such structural dynamics play in HCN allostery. To gain insight into dimer structural dynamics, and considering that the transient nature of the IR dimer intermediates makes them challenging to isolate experimentally, we performed MD simulations of HCN4 IR dimer constructs with varying numbers of bound cAMP molecules (Figure 1b,c and Table S1), using a protocol similar to that implemented for

tetramer symmetry from 4- to 2-fold via diagonal distortions is sufficient to accommodate two adjacent inactive CBDs, whereas dissociation of the tetramer into dimers permits accommodation of a full set of four inactive CBDs.43 Notably, both of these pathways displayed a dimeric organization of structural transitions, suggesting that IR dimers play a role in HCN allostery.43 Furthermore, sedimentation equilibrium data for the isolated HCN IR,3,17,44 electrophysiology of HCN binding-site mutants,45 and functional fluorometry experiments46 have demonstrated that HCN activation proceeds via a pair of allosterically coupled IR dimers, whereby pairs of IR protomers D

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MD Simulation Protocol. All MD simulations were performed in triplicate with NAMD 2.9 software49 on the Shared Hierarchical Academic Research Computing Network (SHARCNET),50 using the CHARMM27 force field and following the protocol implemented previously.42 A summary of the MD simulations performed for the dimer constructs is given in Table S1. Analysis of HCN4 IR Structural Dynamics. All dimer simulations were analyzed through backbone root-mean-square deviations (RMSDs) of key IR structural elements (Figure 1b) from the active-state structure (represented by the 3OTF structure, as established previously;3,5,51 Figures 2, 4, S2 and S6), backbone N−H order parameters (S2; Figures S3 and S4), backbone root-mean-square fluctuations (RMSFs; Figures 3 and 5), and RMSD-based active-versus-inactive structure similarity measures (SMs; Figures 6, 7, and S5), similar to the analyses of the previous monomer and tetramer simulations.42 The RMSDs and SMs were used to assess the propensities of the major IR structural components for activelike (i.e., resembling/approaching the 3OTF structure; Figures 1b and S1a) versus inactive-like (i.e., resembling/approaching the average structure derived from PDB entry “2MNG”, which was solved for an HCN4 IR construct trapped in an inactive conformational state;19 Figure S1b) structural arrangements, where SM values approaching 1 or −1 indicate protein conformations with active- or inactive-like structural arrangements, respectively.42 Meanwhile, the backbone N−H order parameters and RMSFs quantify amplitudes of local and longrange structural fluctuations, respectively. All analyses were performed following a protocol similar to that implemented for the previous monomer and tetramer simulations42 (see Supporting Information text for additional details), averaging the data across the three simulation replicates. However, whereas the tetramer data examined previously42 were also averaged across the constituent protomers of each tetramer, the two constituent protomers of each dimer were assessed individually due to the inherent differences in the interprotomer contacts formed by each of the two protomers (Figure 1b; compare to Figure 1a). Comparisons of the fully apo and fully holo dimers with the respective monomer and tetramer simulations42 (Figure 1c, left and right panels) were performed to assess the effects of IR dimerization on protomer structural dynamics, whereas comparisons of the P1 holo/P2 apo and P1 apo/P2 holo singly bound dimers with the fully apo and fully holo dimers (Figure 1c, middle panels) were performed to assess dimer structural dynamics potentially relevant for interprotomer allostery.

our examination of IR monomers and tetramers.42 In particular, previous simulations of dimer constructs lacking bound cAMP (“fully apo” dimer; Table S1 and Figure 1c, left panel) were complemented here by simulations of dimers with cAMP bound to both constituent protomers (“fully holo” dimer; Table S1 and Figure 1c, right panel), and compared to the respective monomer and tetramer simulations from our previous work42 to assess the effects of IR dimerization on protomer structural dynamics in the apo and cAMP-bound states. In addition, simulations of dimer constructs with cAMP bound to only one of the two constituent protomers (“P1 holo/P2 apo” and “P1 apo/P2 holo” dimers; Table S1 and Figure 1c, middle panels) were implemented and compared to the fully apo and fully holo dimer simulations to assess variations in dimer structural dynamics that underlie interprotomer allostery, such as the cooperativity of cAMP binding to HCN reported previously based on functional fluorometry data.46 The simulation of each system included three 100 ns trajectories (Table S1). Our findings not only corroborate the notion that both the monomeric and dimeric states are capable of accommodating inactive CBD conformations that are incompatible with the tetrameric state,43 but more notably, they unexpectedly reveal that the dimeric state confers a sensitization of the IR to cAMP-modulated allostery affecting both the TD and CBD components of the IR.



METHODS Overview. Molecular dynamics (MD) simulations in explicit solvent were performed for dimers of the IR from human HCN4, containing the full TD and CBD regions of HCN4 (Figure 1b). The simulations were set up and executed following a protocol previously validated for monomer and tetramer simulations of the HCN4 IR,42 with initial structures for the simulations of all four dimers (Figure 1c and Table S1) constructed starting from the X-ray crystal structure of a single IR protomer (monomer) bound to a single cAMP molecule (PDB ID 3OTF).5 Details about the preparation of the initial structures as well as the MD simulation protocols and analyses are described below. Molecular Dynamics Simulation Protocol. Initial Structure Preparation. The HCN4 IR construct spanning residues 521−717 of the HCN4 intracellular region was used for all MD simulations. An initial cAMP-bound protomer structure was obtained from the X-ray crystal structure of the cAMP-bound IR (PDB ID 3OTF),5 as previously done for the IR monomer.42 The corresponding tetramer structure (Figure 1a) was then obtained by using Swiss PDBViewer47 to generate four copies of the monomer structure by applying “BIOMT” rotation/translation structure transformations specified in the header lines of the 3OTF PBD text file. Finally, the initial structure for the fully holo dimer was obtained by deleting one pair of adjacent protomers from the tetramer structure (Figure 1a,b), using Swiss PDBViewer47 to confirm that the two remaining protomers were adjacent to, and thus interfaced with, one another (Figure 1b). The initial structures for the fully apo, P1 holo/P2 apo, and P1 apo/P2 holo dimers were subsequently obtained from the initial structure for the fully holo dimer simply by deleting one or both of the bound cAMP molecules (Figure 1b,c and Table S1). The remaining steps of the structure setup, including addition of hydrogen atoms and set up of the structure topology parameters and explicit solvent, were performed with VMD 1.8.648 as described previously.42



RESULTS Overall Structural Dynamics. As an initial assessment of protomer structural dynamics, RMSDs of the full IR and its constituent domains (i.e., TD and CBD) from the active-state IR structure (represented by the cAMP-bound structure)3,5,51 were computed for the constituent protomers of the fully apo and fully holo dimers and compared to the respective monomer and tetramer data42 (Figures 2a−d and S2a,b). For the full IR, the RMSD values exhibit an overall decreasing trend in the order monomer > dimer > tetramer for both the apo and holo states (Figure 2a,b), suggesting that a partial stabilization of the active-state IR structure occurs upon dimerization. This trend is particularly prominent for the TD region of the IR (Figure 2c,d). For the CBD region, however, the RMSDs exhibit ranges of values comparable to those of the respective monomers, with E

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Figure 4. (a, c, e, g) Distributions of backbone root-mean-square deviations (RMSDs) of the full IR and the tetramerization domain (TD) from their configurations in the initial 3OTF structure, as observed for protomer 1 of the fully apo dimer (red), P1 holo/P2 apo dimer (orange), P1 apo/P2 holo dimer (blue), and fully holo dimer (green) of the HCN4 IR. (a) RMSDs for the entire HCN4 IR, (c) RMSDs for the TD, (e) RMSDs within the αA′−αB′ segment of the TD, (g) RMSDs within the αC′−αD′ segment of the TD. (b, d, f, h) As in (a, c, e, g), but for protomer 2 of each dimer (schematically outlined in the inset panel). The statistics are reported as in Figure 2. F

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Figure 5. (a) Backbone root-mean-square fluctuations (RMSFs) vs residue number plots for protomer 1 of the fully apo dimer (red), P1 holo/P2 apo dimer (orange), P1 apo/P2 holo dimer (blue), and fully holo dimer (green) states of the HCN4 IR (schematically outlined in the inset panel), averaged across all replicates for the respective states. (b) As in (a), but for protomer 2 of each dimer. Other details are indicated as in Figure 3.

smaller values observed for the holo states (Figure S2a,b), suggesting that as with the monomers,42 active-state CBD structure stabilization in the dimers is controlled mainly by cAMP binding rather than IR self-association. TD Structural Dynamics in Fully Apo and Fully Holo IR Dimers. The TD structural dynamics in the fully apo and fully holo dimers were further examined by computing RMSDs of the major α-helical components of the TD (i.e., the αA′−αB′ and αC′−αD′ regions; Figure 1b) for the constituent protomers (Figure 2e−h), along with backbone N−H order parameters (S2; Figure S3) and root-mean-square fluctuations (RMSFs; Figure 3). The αA′−αB′ region RMSDs for both protomers of the fully apo dimer demonstrate an extent of

deviation from the active-state conformation that is intermediate between those for the apo/monomer and apo/ tetramer (Figure 2e), suggesting that the protomer αA′−αB′ regions experience a partial stabilization of their active-state conformations in the dimer relative to the monomer. This stabilization is further reflected by a concurrent quenching of local structural dynamics within the protomer αA′−αB′ regions, as reflected by higher S2 values for the dimer than for the monomer (Figure S3a). In addition, protomer 2 experiences a quenching of long-range αA′−αB′ region dynamics relative to the CBD which is similar to that observed for the apo/tetramer, as reflected by the αA′−αB′ region RMSFs for protomer 2 (Figure 3a, green vs black plots). G

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Figure 6. Two-dimensional plots of the computed active-versus-inactive structure similarity measures (SMs) for the N3A (i.e., αE′−αA) and αB−αC regions of the CBD, as observed in the context of the apo/monomer,42 fully apo dimer, and apo/tetramer42 states of the HCN4 IR (schematically outlined in the inset panel). (a, b) Apo/monomer (brown), protomer 1 of the fully apo dimer (red), and apo/tetramer (black); (c, d) apo/monomer (brown), protomer 2 of the fully apo dimer (green), and apo/tetramer (black). Note that each left panel (a, b) includes the same data but with a different order of front vs back layers. Similarly, each right panel (c, d) includes the same data, again with different orders of front vs back layers. The CBD structural topologies (i.e., “inactive” vs “active”) represented by the quadrants of each graph are indicated. For comparison, the boundaries of the range of similarity measure values computed from the individual structures of the inactive-state structure ensemble (PDB ID 2MNG) are also indicated (red dashed lines).

Although the involvement of the protomer 2 αA′−αB′ region in the interprotomer interface of the dimer was expected to stabilize the protomer 2 αA′−αB′ region, as was previously observed for the apo/tetramer,42 the partial stabilization observed for the protomer 1 αA′−αB′ region was unexpected due to the absence of direct elbow−shoulder interprotomer interactions involving this region (Figure 1a,b). In contrast, the αC′−αD′ region RMSDs for the fully apo dimer demonstrate a range of values comparable to the apo/tetramer for protomer 1, but comparable to the apo/monomer for protomer 2 (Figure 2g). Because interprotomer interactions with the αC′−αD′ region are present for protomer 1, but absent for protomer 2 (Figure 1b), these RMSDs suggest that stabilization of the active-state conformation of the αC′−αD′ region depends mainly on participation in a direct elbow−shoulder interprotomer interface. Compared to the fully apo dimer, the αA′−αB′ region RMSDs for both protomers of the fully holo dimer demonstrate a decrease to values comparable to those for the tetrameric states (Figure 2e,f). In addition, a visible quenching of long-range αA′−αB′ region dynamics relative to the CBD is observed for protomer 1 in the fully holo dimer relative to the fully apo dimer, as evidenced by a visible decrease in the αA′−αB′ region RMSFs for protomer 1 (Figure 3a,b, red plots) and an accompanying decrease in the TD region RMSDs for protomer 1 (Figure 2c,d, red plots). Therefore, cAMP binding to the dimer appears to promote further stabilization of the active-state structural arrangement of the αA′−αB′ region, i.e., beyond the stabilization observed for the fully apo dimer

relative to the apo/monomer. Conversely, the αC′−αD′ region RMSDs for protomer 2 demonstrate an unexpected increase compared to the fully apo dimer (Figure 2g,h, green plots), with an accompanying decrease in αC′−αD′ region S2 values (Figure S3, green plots). Thus, cAMP binding to the dimer appears to promote an enhancement of dynamics within the αC′−αD′ region of protomer 2. Interestingly, these apo-versusholo differences observed for the dimeric state were absent or less prominent in the monomeric and tetrameric states,42 suggesting that the dimeric state sensitizes the TD to perturbation by cAMP by amplifying the allosteric coupling between cAMP binding to the CBD and dynamics of the TD region, the latter of which are critical for tetramerization. However, the comparative analyses of the fully apo and fully holo dimers are not informative on the interprotomer allostery within the dimer, which is another functionally critical and experimentally testable attribute of HCN regulation. Thus, to investigate interprotomer allostery in the dimer, we simulated dimers with partial cAMP occupancies. TD Structural Dynamics in IR Dimers with One Bound cAMP. The analyses performed for the fully apo and fully holo dimers were then extended to the singly bound P1 holo/P2 apo and P1 apo/P2 holo dimers (Figure 1c middle panels, and Table S1), as shown in Figures 4, 5 and S4, with the goal of assessing whether TD structural dynamics are influenced by interprotomer allostery. One of the most notable features of Figure 4 is that the αA′−αB′ region RMSDs for protomer 1 decrease to values comparable to those for the fully holo dimer only when cAMP is bound to protomer 1 (Figure 4e, orange H

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Figure 7. (a, b) Two-dimensional plots of the computed active-versus-inactive structure similarity measures (SMs) for the N3A (i.e., αE′−αA) and αB−αC regions of the CBD, as observed for protomer 1 of the fully apo dimer (red), P1 holo/P2 apo dimer (orange), P1 apo/P2 holo dimer (blue), and fully holo dimer (green) states of the HCN4 IR (schematically outlined in the inset panel). (c, d) As in (a, b), but for protomer 2 of each dimer. Other labels and symbols are as in Figure 6.

and green plots). However, a decrease in the αA′−αB′ region RMSFs for protomer 1 (compared to the fully apo dimer; Figure 5a) is observed when cAMP is bound to either protomer 1 or protomer 2. Furthermore, the αA′−αB′ region RMSDs for protomer 2 remain at values comparable to the fully apo dimer for both singly bound dimers, with a full decrease to values comparable to the tetrameric states occurring only in the fully holo dimer (Figure 4f). Meanwhile, the αC′−αD′ region RMSDs for protomer 2 demonstrate a considerable increase, compared to the fully apo dimer, whenever cAMP is bound to protomer 2 (Figure 4h, blue and green plots). However, smaller increases in the protomer 2 αC′−αD′ region RMSDs are also observed upon cAMP binding to protomer 1, regardless of whether or not cAMP is already bound to protomer 2 (Figure 4h, orange vs red plots and green vs blue plots). In addition, cAMP binding to protomer 1 produces a visible decrease in protomer 2 αC′−αD′ region S2 values when protomer 2 is cAMP-bound (Figure S4b, green vs blue plots) and a visible decrease in protomer 2 αC′−αD′ region and αB′−αC′ loop S2 values when protomer 2 is cAMP-free (Figure S4b, orange vs red plots), highlighting enhancements of local dynamics within these regions. Interestingly, the latter perturbation is accompanied by an increase in long-range αA′−αB′ region dynamics for protomer 2, as evidenced by an increase in both αA′−αB′ region RMSFs and TD region RMSDs for protomer 2 (Figures 4d and 5b, orange vs red plots), suggesting the emergence of an αA′−αB′/ CBD relative movement via a hinging motion around the protomer 2 αC′−αD′ region, similar to the hinging motion observed previously upon dissociation of the apo tetramer into dimers.43 Furthermore, the enhancements of local αB′−αC′ loop and long-range αA′−αB′ region dynamics in protomer 2

are reversed by subsequent cAMP binding to protomer 2 (Figures 4d, 5b, and S4b, green vs orange plots). Together, the comparative analyses of TD structural dynamics for fully apo, fully holo, and singly bound dimers (Figures 4, 5, and S4) suggest that in the dimeric state, cAMP binding to either protomer within the dimer perturbs the TD region of not only the “host” binding protomer but also the adjacent protomer, suggesting that TD structural dynamics are influenced significantly by interprotomer allostery within the dimeric state. To investigate whether a similar pattern of interprotomer allostery applies to the CBD region as well, we extended our analyses to the CBD structural dynamics, which involve transitions between active and inactive CBD conformations. CBD Structural Dynamics in Fully Apo and Fully Holo IR Dimers. The CBD structural dynamics in the fully apo and fully holo dimers were examined by computing active-versusinactive similarity measures (SMs) for the protomer CBDs of the fully apo and fully holo dimers, using RMSDs from the experimental active and inactive conformations for the αB−αC helices and N3A (Figure S1) as described previously42 (see Methods and Supporting Information text), and compared to the respective monomer and tetramer data from our previous work42 (Figures 6 and S5). The propensity of the CBDs for active-like conformations that was observed for the holo/ monomer and holo/tetramer42 is preserved in the fully holo dimer, as indicated by distributions of SM values overlapping those observed for the holo/monomer and holo/tetramer (Figure S5) and by RMSDs computed for the CBDs and their major α-helical components (i.e., the PBC, αB−αC, and N3A; Figure 1b) that are comparable to those for the holo/monomer and holo/tetramer (Figure S2b,d,f,h). Meanwhile, as with the apo/monomer, the fully apo dimer exhibits a stronger I

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Figure 8. Schematic summary of the observed changes in protomer structural dynamics that occur among the simulated dimeric (b, e, g, h), monomeric42 (a, d), and tetrameric42 (c, f) states of the HCN4 IR. The two constituent protomers of each dimer (i.e., P1 and P2) are indicated, along with key structural elements of each protomer: the αA′−αB′ (“A′−B′”), αB′−αC′ connecting loop (“B′−C′ loop”) and αC′−αD′ (“C′−D′”) segments of the TD; and the CBD domain (“CBD”). Variations in the local structuring of each TD segment relative to the apo/monomer, as assessed by structural-region-specific RMSD values and S2 order parameters, are indicated as follows: blue = active-structure stabilization; cyan = partial active-structure stabilization; red = enhancement of structural dynamics. Dashed lines/rectangles denote unstructured or partially structured TD segments, whereas solid lines/rectangles denote structured TD segments. Variations in the long-range dynamics of intraprotomer αA′−αB′ region movement relative to the CBD, as gauged relative to the apo/monomer by protomer RMSFs, are indicated as follows: blue double arrow = quenched dynamics; cyan double arrow = partially quenched dynamics; black double arrow = dynamics comparable to the apo/monomer. Finally, variations in the conformational propensities of the CBDs, as assessed by the SM value distributions, are indicated as follows: circle = mainly inactivelike conformations (similar to the apo/monomer); square = mainly active-like conformations (similar to the tetramers); outline thickness qualitatively illustrates the relative degree of preference for inactive-like (circle) or active-like (square) conformations, in the order thick line > medium line > thin line; empty circle/square = apo CBD; gray-filled circle/square = cAMP-bound CBD.

only, protomer 1 demonstrates a greater propensity for inactive-like CBD conformations than in the fully apo dimer, as indicated by an increased population of SM values entering or approaching the range of values defined by the inactive-state structure ensemble (Figure 7a,b, blue vs red plots). Therefore, our simulations lead to the notable result that the inactive-toactive conformational transitions for the dimer CBDs are coupled to one another in a markedly and unexpectedly directional manner: binding of the first cAMP molecule to the protomer 1 CBD promotes positive interprotomer cooperativity, whereas binding of the first cAMP molecule to the protomer 2 CBD promotes negative interprotomer cooperativity.

propensity for inactive-like CBD conformations than the apo/ tetramer, as indicated by dimer SM values entering or approaching the range of values defined by the previously solved inactive-state structure ensemble19 (Figure 6). Indeed, RMSDs computed for the CBDs and their major α-helical components highlight a visible inactive-to-active transition of the monomer and dimer CBDs upon cAMP binding (Figure S2b,d,f,h vs a,c,e,g), suggesting that similar to the monomers, the dimers exhibit CBD structural behavior that is dependent mainly on cAMP binding. CBD Structural Dynamics in IR Dimers with One Bound cAMP. As a further assessment of how dimer CBD structural dynamics depend on cAMP binding, the analyses performed for the protomer CBDs of the fully apo and fully holo dimers were extended to the singly bound P1 holo/P2 apo and P1 apo/P2 holo dimers (Figure 1c middle panels and Table S1). Notably, when cAMP is bound to protomer 1 only, protomer 2 demonstrates a visibly lower propensity for inactive-like CBD conformations than in the fully apo dimer, as indicated by a reduced population of SM values entering or approaching the range of values defined by the previously solved inactive-state structure ensemble19 (Figure 7c,d, orange vs red plots). Conversely, when cAMP is bound to protomer 2



DISCUSSION The key findings from the simulations reported here are summarized in Figure 8. Our results not only corroborate the notion that both the monomeric and dimeric states are capable of accommodating inactive CBD conformations that cannot be accommodated in a holo-like tetramer with 4-fold symmetry (Figures 6 and 8a−c),43 but also reveal functionally relevant attributes of HCN4 IR dynamics that have so far remained elusive. Notably, our simulations show that dimerization “fineJ

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Figure 9. Modeling dimers of dimers in the context of HCN IR tetramer assembly. (a−f) Outline of proposed contributions of dimer allostery to tetramer assembly in intact HCN, based on findings from our previous43 and current work. Dimer (a−c) and tetramer (d−f) structures are schematically represented in a manner similar to Figure 8. The transparent dark green rectangles between the two dimers in (c) indicate an enhanced favorability of interprotomer interactions between the dimers (relative to the fully apo state), whereas the transparent light green rectangles between the two dimers in (b) indicate a partially enhanced favorability of interprotomer interactions between the dimers. The relative favorability of interdimer interactions is also qualitatively represented by the directionalities of the vertical equilibrium arrows between the dimer and tetramer structures. (g−j) Hypothesis about the relevance of the interprotomer “breathing” motion that was previously observed within the HCN IR tetramer.53,54 (g, h) Schematic representation of the previously observed breathing motion,53,54 highlighting the alternation between 4-fold and dimer-of-dimers symmetries that is expected to occur as a result of the breathing movement. (i, j) Schematic outline of one of the structural transitions observed from our previous hybrid-tetramer simulations,43 whereby a tetramer with four inactive CBDs (circles) undergoes inactive-toactive conformational shifts in two adjacent CBDs (squares), along with a diagonal distortion of the tetramer along the normal modes of the breathing motion53,54 (g, h), to relieve CBD/TD steric clashes with the inactive CBDs (gray starbursts). (i, j) Adapted from VanSchouwen and Melacini (2016).43 K

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disordered monomers or more structured higher oligomers, which is something beyond the scope of what could be predicted by the Monod−Wyman−Changeux model of allostery. Third, examination of TD structural dynamics in dimers with only one bound cAMP (Figures 4, 5, and S4) revealed that cAMP binding to either protomer within the dimer can perturb the TD region of not only the host binding protomer but also the adjacent protomer (Figure 8b,e,g,h). This latter observation suggests that TD structural dynamics are influenced significantly by interprotomer allostery within the dimeric state. In particular, cAMP binding to protomer 1 produces enhancements of local αC′−αD′ region and αB′−αC′ loop dynamics and long-range αA′−αB′ region dynamics in protomer 2, whereas cAMP binding to protomer 2 produces a quenching of long-range αA′−αB′ region dynamics in protomer 1 (Figure 8b,e,g,h). Therefore, the interprotomer allostery observed for cAMP-modulated perturbations of CBD structural dynamics also appears to influence TD structural dynamics in the dimeric state, providing a further contribution to the allosteric sensitization of the TD that is conferred by dimerization. Our observation that the dimeric state sensitizes the protein to cAMP-modulated allosteric perturbations (Figures 8 and 9a−c) is also consistent with a recent bioinformatics study by Bergendahl and Marsh (2017),52 which found that proteins that self-assemble into quaternary structures with dimeric symmetry, including dimers and dimers of dimers, demonstrate an enhancement of allosteric function compared to proteins with monomeric or 4-fold-symmetrical multimeric structures. More notably, such dimer-specific allosteric “enrichment” may also provide an explanation for the relevance of the interprotomer “breathing” motion that was previously proposed for the HCN IR tetramer (Figure 9g,h).53,54 Specifically, the interprotomer breathing may serve to transiently establish a dimer-of-dimers symmetry within the tetrameric state (Figure 9h), thereby contributing to interprotomer allostery by allowing dimer-like allostery to transiently manifest within the tetramer. Indeed, our previous hybrid-tetramers simulation work43 demonstrated that when tetramers with four inactive CBDs underwent inactive-to-active CBD conformational shifts to relieve the CBD/TD steric clashes, the result was always two or four adjacent active CBDs, as would be expected in the presence of positive intradimer cooperativity,45,46 concurrent with an accompanying diagonal distortion of the tetramer along the normal modes of the breathing motion53,54 (Figure 9i−j vs Figure 9g−h), suggesting the possibility that these two phenomena are linked to one another. Furthermore, we found that intradimer allostery allows cAMP binding to protomer 1 to produce a transient enhancement of αA′−αB′/CBD relative movement (Figures 5b and S4b, orange vs red plots; Figure 8g vs Figure 8b,e), which demonstrates similarity to the enhanced movement observed previously upon dissociation of the apo tetramer into dimers,43 hinting that the modulation of IR structural dynamics by dimerization may also contribute an embedded means for facilitating reversible tetramer assembly from dimers. Thus, although partly speculative, these considerations suggest that by promoting dimer-like allostery, the interprotomer breathing may facilitate cooperative protomer conformational transitions as well as reversibility of tetramer formation.

tunes” IR dynamics to enhance the allosteric response of the HCN4 IR to cAMP. Such “fine-tuning” is absent in the monomeric IR, where the TD flexibility reduces the coupling with the CBD, and in the tetrameric IR, where tight structural packing minimizes cAMP-induced changes. The dimer-specific sensitization to cAMP-modulated allostery that is revealed here includes three key features that affect both the CBD and TD regions of the IR. First, dimers with only one bound cAMP have shed light on the nature of the positive intradimer cooperativity of cAMP binding identified from previous functional fluorometry electrophysiology on intact HCN.46 Specifically, similarity measure (SM) distributions suggest that when cAMP binds to the protomer 1 CBD first, the protomer 2 CBD undergoes a shift toward less inactive conformations (Figure 7), facilitating subsequent binding of cAMP to protomer 2 (Figure 8b,e,g), whereas the opposite trend occurs when cAMP binds to the protomer 2 CBD first (Figures 7 and 8b,e,h). Therefore, the positive intradimer cooperativity is highly asymmetric and follows a specific protomer-to-protomer directionality of successive cAMP binding events and associated inactive-toactive conformational shifts within the CBDs of the HCN dimer (Figure 8b,e,g). These results provide a viable rationalization for the observation based on confocal patch-clamp fluorometry that positive intradimer cooperativity is concurrent with negative interdimer cooperativity. Second, in addition to influencing allostery at the level of the CBD and cAMP binding, the dimeric state also appears to sensitize the TD to cAMP-associated perturbations, providing a previously unknown contribution to allosteric control of the TD (Figure 8a−f). Examination of TD structural dynamics in fully apo and fully cAMP-bound dimers (Figures 2, 3, and S3) revealed that in addition to an expected stabilization toward active-like structure for the TD segments that participate in the interprotomer elbow−shoulder interface of the dimer (i.e., the protomer 1 αC′−αD′ and protomer 2 αA′−αB′ regions; Figure 1b), dimerization of the HCN IR produces an unexpected local stabilization of the protomer 1 αA′−αB′ region despite its lack of direct participation in interprotomer elbow−shoulder interactions (Figure 8a,b,d,e). More notably, cAMP binding to the dimer was found to produce a stabilization of the activestate structural arrangement of the αA′−αB′ region of protomer 1 and a visible enhancement of local dynamics within the αC′−αD′ region of protomer 2 (Figure 8a−f). Given that cAMP binding is known to promote tetramer formation,3,15−17 these cAMP-associated perturbations may facilitate tetramer formation in intact HCN by shifting the affected TD structural elements toward a structural state that is more conducive to interprotomer interactions (Figure 9a−f). However, such perturbations were absent or less prominent upon cAMP binding to the monomer or tetramer,42 suggesting that the dimeric state modulates the allosteric coupling between cAMP binding to the CBD, and dynamics of the TD region that are critical for tetramerization, thus enhancing the allosteric response of the TD to cAMP binding. This phenomenon may arise from the TD being less unstructured in the dimer than in the monomer, yet not as rigid as in the tetramer. The flexibility of the monomer allosterically decouples the TD from the CBD, whereas the structural packing of the tetramer reduces the amount of room for TD perturbation in response to cAMP. These results suggest that partial self-association of intrinsically unstructured proteins is an effective means to introduce allosteric couplings that would otherwise be absent in more L

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Notes

CONCLUSIONS The dimer simulations presented here have provided unprecedented insight into the HCN IR dimers and their role in HCN allostery. First, the dimeric state sensitizes the IR to cAMP-modulated allosteric perturbations that affect the structural dynamics of both the CBD and TD domains. Such cAMP-dependent TD perturbations were absent or less prominent in the monomeric and tetrameric states, suggesting that the dimeric state enhances the allosteric coupling between cAMP binding to the CBD, and dynamics of the TD region that are critical for tetramerization, thus ensuring an efficient allosteric response. Second, interprotomer cooperativity of CBD conformational shifts promotes a positive intradimer cooperativity of cAMP binding that follows a specific protomerto-protomer directionality within the HCN dimer and rationalizes previous fluorometry and electrophysiological data.45,46 Finally, interprotomer allostery may also rationalize the functional relevance of the interprotomer breathing motion that was previously proposed for the IR tetramer,53,54 whereby dimer-like allostery can manifest via transient dimer-of-dimers symmetry within the tetrameric state.



The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors thank Professors W.N. Zagotta (U. of Washington), E. Accili (UBC), A. Moroni (U. Milan), S.S. Taylor (UCSD), and M. Akimoto (Keio U.), as well as S. Boulton and K. Moleschi (McMaster), for helpful discussions. G.M. received funding from Canadian Institutes of Health Research (Grant MOP-68897) and Natural Sciences and Engineering Research Council of Canada (Grant RGPIN2014-04514).



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b10125. Additional details of the methodology for the analyses performed on the MD simulations; summary of the MD simulations performed for the HCN4 IR dimers (Table S1); comparison of the previously solved active cAMPbound (3OTF) and inactive apo-state (2MNG) structures of the HCN4 cAMP-binding domain (CBD) (Figure S1); distributions of backbone root-mean-square deviations (RMSDs) of the CBD and its major α-helical structural elements from their configurations in the initial 3OTF structure, as observed in the context of the apo and holo monomers, dimers, and tetramers of the HCN4 IR (Figure S2); backbone N−H order parameters (S2) versus residue number plots for the apo and holo monomers, dimers, and tetramers of the HCN4 IR (Figure S3); backbone N−H order parameters (S2) versus residue number plots for the fully apo, P1 holo/P2 apo, P1 apo/P2 holo, and fully holo dimers of the HCN4 IR (Figure S4); two-dimensional plots of the computed active-versus-inactive structure similarity measures (SMs) for the N3A (i.e., αE′−αA) and αB−αC regions, as observed in the context of the holo monomer, dimer, and tetramer states of the HCN4 IR (Figure S5); distributions of backbone root-mean-square deviations (RMSDs) of the CBD and its major α-helical structural elements from their configurations in the initial 3OTF structure, as observed for the fully apo, P1 holo/P2 apo, P1 apo/P2 holo, and fully holo dimers of the HCN4 IR (Figure S6) (PDF)



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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (905) 525-9140 x26959. Fax: (905) 522-2509. ORCID

Giuseppe Melacini: 0000-0003-1164-2853 M

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