The structural mechanism underlying the antithetic effect of homologous RND1 and RhoD GTPases in plexin regulation

Plexins are semaphorin receptors that play essential roles in neuronal axon guidance and in many other important biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism, and is modulated by small signaling GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations we showed that RND1 reinforces plexin dimerization interface whereas RhoD destabilizes it due to their differential interaction with cell membrane. Upon binding plexin dimers at the Rho-GTPase binding (RBD) domains, RND1 and RhoD interact differently with the inner leaflet of cell membrane, and exert opposite effects on the dimerization interface via an allosteric network involving the RBD domain, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively-charged membrane interface.


Introduction
Plexins are a family of nine single-pass transmembrane receptor proteins including plexin A1-4, B1- The RBD-GTPase binding is integral to plexin regulation from the intracellular environment. Overexpression of Rac1 leads to higher cell surface expression of plexin and enhances plexin interaction with semaphorin, suggesting that Rac1 acts as an upstream activator of plexin (Vikis and H., 2002).
Moreover, binding of over-expressed RND1 to plexin triggers cell collapse in the absence of semaphorin, suggesting that RND1 is a more potent activator than Rac1 for plexin (Zanata et al., 2002).
Simultaneous extracellular binding of semaphorin and RBD-GTPase appears to be a prerequisite for full activation of at least some plexins (Bell et al., 2011), but RBD binding with some other Rho GTPases appears to attenuate plexin activity. RhoD binds plexin with similar affinity as RND1 (Fansa et al., 2013), presumably in the same mode as Rac1 and RND1, in sharp contrast it strongly inhibits To understand the apparent paradox regarding the antithetic effects of RhoD and other RhoGTPase on plexin activation, we determined the crystal structure of the RhoD/plexin B2-RBD complex, which confirmed that the binding mode of RhoD to RBD is similar to that of other RhoGTPases. Furthermore, we here modeled and simulated RND1 and RhoD complexes with plexin A4 (Figure 1C), to investigate the structural mechanisms underlying RND1 as an activator and RhoD as an attenuator in plexin regulation. The simulations showed that RND1 binding is compatible with the dimerization of plexin A4 while RhoD binding is likely disruptive to the dimerization. The simulations revealed two distinct modes of interactions of RND1 and RhoD with the membrane: RND1 interacts with the membrane loosely and its long C-terminal tail serves as a flexible tether from the membrane ( Figure 1D), whereas RhoD interacts with the membrane in a specific manner using an electrostatically-positive membranecontacting interface ( Figure 1E) that is absent in RND1. As a result, RND1 binding strengthens plexin dimerization by stabilizing the RBD, RhoD interacts with the membrane closely and presents a hindrance to plexin dimerization.

Crystal structure of the complex between RhoD and the plexin B2-RBD domain
Complex structures of the RBD domain with plexin activators such as RND1 or Rac1 have been previously resolved. To experimentally determine the binding mode between plexin and RhoD, a negative plexin regulator, we screened various combinations of RhoD and the intracellular region of plexin family members from different species for crystallization, which resulted in crystals of the complex of mouse plexin B2 and human RhoD bound to the GTP analogue GMP-PNP. Analyses of the diffraction data suggested that plexin B2 degraded during the incubation in crystallization drops, and the crystals only contained the complex between RhoD and the RBD of plexin B2. We solved the structure to 3.1 Å resolution by molecular replacement (Supplemental Table 1). See methods for details). The asymmetric unit of the crystal contains two RhoD molecules, each of which binds to one plexin B2 RBD molecule. Surprisingly, the two RBD domains form a domain-swapped dimer in the structure, with the N-terminal portion of one molecule fold together with the C-terminal portion of the other (Supplemental Figure 1). This domain-swapped dimer is likely a crystallization artifact because it cannot form in intact plexin. We therefore consider each RBD domain formed by the two halves of the two molecules as a representative of one intact, unswapped RBD, as its conformation is very similar to the structures of other RBDs ( The GTP analogue GMP-PNP and Mg 2+ together stabilize the ligand-binding switch I and switch II regions in the active conformation, which make an extensive interface with one side of the beta-sheet of the RBD (Figure 2). All the residues in RhoD involved in interacting with the RBD are identical between human and mouse RhoD, suggesting that the cross-species complex that we crystallized is a valid representative of the RhoD/plexin complex. Interestingly, a superimposition of the RhoD/Plexin B2-RBD complex with the RND1/Plexin B1-RBD complex based on the RBD domains shows that the orientation of RhoD and RND1 relative to the RBD domains are slightly different ( Figure 2). Compared with that in RND1, the switch II helix in RhoD is placed further away from the RBD, which appears to be required to accommodate Phe85, which is larger than Cys81, the corresponding residue in RND1.
This difference leads to different pivots of the two GTPases relative to the RBD, which propagates to a larger difference in the opposite side of the molecule where the insert helices (αi), a unique helical segment present in the Rho subfamily but not other GTPases, is located ( Figure 2). In the context of the active dimer of full-length plexin on the plasma membrane, the area near αi of the RhoGTPases is placed to face the membrane surface. This orientational difference between RhoD and RND1 relative to plexin therefore may influence their interactions with the membrane, as analyzed in detail below. It is unclear how the subtle difference in orientation confers the opposite regulatory effects between RND1 and RhoD. To shed further light on this question, we performed further investigation using MD simulations of Rho GTPase-bound plexin systems.

RhoD and RND1 interacts differently with cell membrane
The membrane may play an important role in plexin regulation by the small GTPases, which are located adjacent to the membrane. Previous studies showed that RhoD does not alter the GAP activity of plexin We simulated the RND1-bound ( Figure 3A) and the RhoD-bound ( Figure 3B) plexin dimers, each for 1 μs three times. In the simulations of the RND1-bound dimer, the amphipathic helices at the C-termini of the RND1 molecules remained anchored to the membrane, and the RND1 linkers between the catalytic domains and the amphipathic helices (residue 189-200) are sufficiently long to not affect the position of the catalytic domains ( Figure 3A). The contact area between the membrane and the catalytic domains remains relatively small, with a mean at approximately 200 Å 2 ( Figure 3D). The two RND1 catalytic domains largely remained in their initial positions, with the root mean square deviation (RMSD) of the Cα atoms with respect to their initial positions fluctuating around 6 Å ( Figure 3E).
The C-terminal tail of the RhoD is shorter and more arginine-rich than the RND1, which is likely membrane-bound and hence restrains the RhoD catalytic domain to the membrane. In contrast to the RND1-bound plexin dimer, in the simulations of the RhoD-bound plexin dimer, the membrane interactions of the RhoD catalytic domains developed extensive and stable interactions with the membrane in the courses of the simulations ( Figure 3D). The contact area of the two RhoD domains with the membrane fluctuated but generally trended upwards. It is apparent that the extent of the membrane interaction is closely correlated with the positioning of the catalytic domains in both the RND1-and the RhoD-bound plexin dimers. With the increase of the membrane interactions, the two RhoD domains deviated substantially from their initial positions, as shown by the RMSD of Cα atoms with respect to their initial positions ( Figure 3E). The RMSD fluctuation of the RhoD domains was larger than the RND1 domains ( Figure 3E), indicating that the differential membrane interactions of RhoD and RND1 lead to differential positioning and dynamics of the two GTPases.
Further analysis showed that RhoD interacts with the membrane with a specific interface involving the insert helix (αi) mentioned above and the α4 helix ( Figure 3C); Arg144, Arg145, His154, and Arg155 in this part of RhoD enjoyed stable interactions with the membrane ( Figure 3F). The RhoD membraneanchoring interface features pronounced positive electrostatic potential that is favorable for membrane interaction ( Figure 3F). In contrast, RND1 interaction with the membrane is much less stable, without a specific membrane interface ( Figure 3F). The positively-charged residues in the RhoD membrane interface are almost all replaced in RND1 ( Figure 3G), and hence the strong electrostatic feature of RhoD in that region is absent in RND1 ( Figure 3F). In conclusion, the tight membrane interaction of RhoD can likely be attributed to the short C-terminal tail and to the positively-charged surface patch, which distinguishes RhoD from RND1.

The differential membrane interactions lead to different RBD position and dynamics
In the plexin dimer, a RhoD or RND1 molecule is located in a space confined by the membrane and the RBD domain and interacts with both simultaneously ( Figure 4A and 4B). In the simulations, the RBD interacts with both RhoD and RND1 stably, although RhoD interacts with RBD with a slightly larger interface than RND1 ( Figure 4C). In addition to RND1-and RhoD-bound plexin dimers, we simulated plexin monomer and dimer with the RBD domains unoccupied, each for 500 ns. We analyzed the positions of the RBD domain with respect to the GAP domain in all our simulations. The RBD domain appeared to be inherently flexible with respect to the GAP domain ( Figure 4E). This is suggested by existing crystal structures of plexins, in which the RBD domain exhibited substantial flexibility with respect to the GAP domain ( Figure 4F). The interface between RBD and GAP appeared to be reduced by the presence of RhoD but not by the presence of RND1 ( Figure 4D). More importantly, the RMSD of the RBD domains with respect to their initial positions were increased by RhoD and decreased by RND1 ( Figure 4E), indicating that RhoD likely displaces the RBD from its native position while RND1 tends to stabilize RBD at that position. Since RBD binds stably with both RND1 and RhoD, the differential RBD positioning and dynamics may likely be attributable to the differential membrane interactions of RND1 and RhoD.

RBD affects plexin dimerization via the buttress segment
The dimerization of plexins is mediated by their dimerization helices that are immediately C-terminal to the juxtamembrane helices ( Figure 1B). The interaction between two dimerization helices in the dimer, which resembles coiled-coil interactions, is reinforced by Helix 11 of the GAP domain ( Figure   1C) (Wang et al., 2013). In crystal structures, Helix 11 is a stable helix, but the segment to its Nterminal is more variable structurally--it takes the form of a 3-10 or an α helix in some crystal structures but in many other structures it is disordered. When it is a 3-10 or an α helix, it becomes an extension of Helix 11 and runs adjacent and in parallel to the dimerization helix, structurally it reinforces the interaction of the two dimerization helices in resemblance to a buttress. Based on this observation we refer to it the buttress segment ( Figure 1A and 1B).
The RBD is connected to the plexin GAP domain by two linkers, a C-terminal and an N-terminal linker.
The C-terminal linker (Residue 1597-1662) is followed immediately by sequentially connected with the buttressing segment. This linker is long and partially disordered in crystal structures, especially in the part closer to the buttressing segment. This suggests that this linker is conformationally highly flexible. The shorter N-terminal linker (residue 1482-1495) connects RBD to the bulk of the GAP domain and is packed against the buttress segment ( Figure 5A and 5B). It is likely that the N and C linkers mediate the regulation of the buttress segment by the RBD since their conformations are expected to be closely coupled with the position of the RBD on one side and with the conformation of the buttress segment on the other.
Our simulations showed that the buttress interaction with the dimerization helix is minimal in a monomeric plexin, and this interaction increases substantially in plexin dimers ( Figure 5D). Importantly, with RhoD binding at the RBD, the buttress interaction with the dimerization helix in the plexin dimer is much reduced compared to that in the RND1-bound dimer or in the dimer where the RBD domains are unoccupied ( Figure 5D), suggesting that RhoD weakens the buttress interaction with the dimerization helix and potentially destabilizes the plexin dimer. In simulations of the RhoD-bound plexin dimer, the buttress segments lost its helical structure and gradually disengaged the dimerization helices ( Figure 5C). In contrast, in simulations of the RND1-bound dimer both the helical structure and the interaction with the dimerization helices are much more stable ( Figure 5B). The difference is reflected by the smaller contact area of the buttress segments and the dimerization helices in the RhoDbound system than in the RND1-bound system ( Figure 5D). Moreover, the simulations showed that in the RhoD-bound dimer the pair of the dimerization helices was conformationally more variable than that in an RBD-unoccupied plexin dimer, and the dimerization helices in an RND1-bound dimer was less variable than the unoccupied dimer ( Figure 5E and 5F). This is consistent with the notion that RhoD binding destabilizes the plexin dimerization interface while RND1 binding may stabilize the dimer.
Based on these simulation results we suggest that the differential membrane interaction of RND1 and RhoD propagates to the plexin dimerization interface and confers antithetic impact to plexin dimerization through the RBD domain and its N and C linkers ( Figure 5B). RhoD binding destabilizes the RBD with respect to the GAP domain, destabilizing the buttress segment with respect to the dimerization interface, and ultimately leads to destabilization of the dimer interface. In contrast, by the same RBD-centered route, RND1 binding helps stabilize the plexin dimer.

Discussion
Plexins function in ways similar to a transistor in that they take two inputs and their responses to the primary input of semaphorin are regulated by the secondary input in form of GTPase binding at the RBD domain. RND1 serves as a promoter of plexin signaling, while RhoD serves as an inhibitor. Our structural and molecular dynamics simulations and analyses suggest that the differential effects of RND1 and RhoD may arise from their differential interactions with the membrane. RND1 interacts with the membrane loosely and non-specifically, while RhoD interacts with the membrane tightly with a specific interface. This difference gives rise to different positioning and dynamics of the RBD domain, which dictates the conformation of the buttress segment adjacent to the dimerization interface of plexin.
We further showed that RhoD binding destabilizes the dimerization interface while RND1 binding helps stabilizes the interface. In short, we reveal an allosteric mechanism that regulates plexin dimerization involving cell membranes, the regulatory GTPases, the RBD domain, and the buttress segment ( Figure 5F).
Our results on RND1 and RhoD offer a framework for the analysis of plexin regulation by Rho GTPases.
We show that the antithetic roles of RND1 and RhoD result from two seemingly minor differences.
First, RhoD furnishes a much shorter C-terminal tail than RND1, and consequently, RhoD is spatially more restrained to the membrane than RND1. Secondly, RhoD features a surface region that is rich of positively-charged residue, which serves as the interface with membranes; these positively charged residues are mutated in RND1. These two differences determine that RND1 and RhoD interact with the membrane differently. We analyzed the sequences of the members of the Rho GTPase family and, to our surprise, we found that these two features are indeed correlated. The Rho GTPases with longer C-terminal tails indeed tend to feature more positively-charged residues at the putative membrane interface ( Figure 6). This suggests that, besides RND1 and RhoD, other Rho GTPases may also be involved in regulations of plexin signaling, and that the GTPases of the Rho family with short C-terminal tails may likely be down-regulators and the other with long C-terminal tails likely upregulators. In cell biology, similar to plexin regulation by Rho GTPases, there are many other cases in which similar protein in the same family interact with their target proteins almost identically yet achieve opposite regulatory effects. Simulations are an expedient platform to gain insight into such mechanisms.
In this study, we chose to focus on plexin A4 as a representative system, despite that crystal structures of the intracellular domains are not available for A4. Unlike A4, for those plexins for which better structural data are available, direct functional data of regulation by RBD-binding of small GTPases are lacking. C1, which is arguably the best structurally characterized plexin in terms of the intracellular domains, is such an example. Even for C1, the structural information is incomplete as the C1/RhoGTPase complex structure is not available. It is thus necessary to construct models from other plexins structures regardless of our choice of plexin system. We resorted to homology modeling (see Methods) to construct A4 structures for simulations, considering the high level of sequence (35% or above overall) and structure similarly among the plexin family members, in particular in the dimer interface. The binding mode between class A plexins and RhoGTPases is particularly highly conserved, as shown by the numerous crystal structures, including that of Plexin B2/RhoD presented in this paper.
We therefore believe the models of the Plexin A4/RND1 and PlexinA4/RhoD complexes are highly reliable. Moreover, our conclusion is not sensitive to the fine structures, as the main driving force is the electrostatic interactions from the interface between the GTPases and membrane ( Figure 3F), rather than any specific residue-residue interactions arising from a specific conformation. Reassuringly, our findings are supported by a recent study on plexin B1, which (Li et al., 2020) identified the functional importance of the buttress segment (or "activation switch loop" as is referred therein) based on analysis of plexin enzymatic turnover, and showed that the segment helps stabilizing the dimerization helix when the plexin active site is occupied by Rap.
Our results suggest that, similar to many other signaling proteins, for plexin the membrane also plays an important role in its regulation. In a membrane environment of a high composition of negativelycharged lipids such as POPS, PIP2, and PIP3, plexin signaling is likely more susceptible to negative regulation by RhoD. There are reports that plexin signaling activates the PI3K/AKT pathway, upon which PIP2 lipids in the membrane are phosphorylated and converted to more negatively charged PIP3 lipids. Our findings raise the question as to whether down-regulation associated with RhoD binding may be part of a negative feedback mechanism for plexin signaling involving the PI3K/AKT pathway.

Supplemental Information
Supplemental information includes the diffraction data and refinement statistics of the crystal structure and orthogonal views of the asymmetric unit of the RhoD/plexin B2-RBD complex crystal.       (B) The number of positively-and negatively-charged residues at the membrane interface. The protein name and the number of residues of its C-terminal tail are marked next to each data point herein.

Construction of the simulation systems
This research included four simulation systems: a plexin monomer, a plexin dimer with RBD unoccupied, an RND1-bound dimer, and a RhoD-bound dimer. In each of these systems, the extracellular domains were absent, but the membrane and the transmembrane helix were included.
Lacking of the crystal structure for plexinA4, we constructed one monomeric structure of the intracellular portion of plexin A4 using homology modelling. The sequence of mouse plexinA4 was taken from NCBI website (http://www.ncbi.nlm.nih.gov/protein). The templates were selected . Modeller generated 100 structural models for the query sequence, and the one with the lowest estimated energy was selected for the construction of our simulation systems.
The plexinA4 dimer structure was obtained from superimposing the monomeric model of plexin A4 onto each protomer of the crystal structure of plexin C1 dimer (PDB entry 4M8N).
We also constructed the complex structure of RND1 with the RBD domain using Modeller 9.17.
All the template structures selected in this research were downloaded from the Protein Data Bank (PDB) database. The templates for constructing RND1 we selected were the resolved crystal structures PlexinA2 hRND1(PDB entry 3Q3J). The process of the Modeller generating structures and the selection standard were the same as used in constructing plexin structure. We separately resolved the RhoD-RBD complex structure using crystallography. This structure was incorporated into the simulation systems.
The CharmmGUI website (http://www.charmm-gui.org/) ( The RND1-bound plexin dimer system was a cubic box of 190 x 190 x 170 Å 3 that contained 679,235 atoms in total. The dimensions of the simulation boxes were chosen so that the minimum distance of any protein in a system was greater than 10 Å to the edge. Na + and Clions were added to maintain physiological salinity (150 mM) and to obtain a neutral charge for the system. The system was parameterized using the CHARMM36 force field (Lee et al., 2016) and TIP3P water model (Jorgensen et al., 1983). The above dimer system with the RBD domains unoccupied system was also used to set up the G-protein-bound dimer system. When the system of two plexin monomer inserted into the membrane was generated, the initial placement of the RND1 molecule bound to RBD of plexin was determined by firstly superimposing one monomer structure in the dimer on the complex of RBD bound with RND1 (PDB entry 3Q3J) with the RBD domains aligned, followed by superimposing one RND1 structure on the RND1 in the complex structure encoded 3Q3J. The RND1 structure was placed at the targeting position. Meanwhile, both the GTP molecule and Magnesium (Mg 2+ ) ion in the complex (PDB entry 3Q3J) were superimposed on the RND1 structure. For the other monomer, the RND1 structure as well as the GTP molecule and Mg 2+ were also placed at the corresponding positions in the same way. Finally, the whole system of the RND1-bound plexin dimer inserted into the membrane was set up. The RND1-bound dimer system was placed in a cubic box of 190 x 190 x 170 Å 3 and 679,235 atoms in total in the system. The RhoD-bound dimer system was a cubic box of 190 x 190 x 170 Å 3 that contained 679,086 atoms in total. Both RhoD and RND1 were GTP-bound in the systems with Mg 2+ coordinating the GTP binding.

MD Simulations
Each initial simulation system was equilibrated under NPT ensemble at 1 bar and 300 K for 5 ns, after energy minimization (50,000 steps) and a preliminary NVT equilibration (500 ps) with the position restraint applied on the heavy atoms of the protein with a force constant of 10 kJ/mol/Å 2 .
Periodic boundary condition (PBC) was imposed on the system to eliminate the boundary effect. A cutoff distance of 12 Å was set for van der Waals interactions and the long-range electrostatic interactions were treated by the Particle Mesh Ewald (PME) method (Darden et al., 1993).

Protein-protein Contact Area Calculation
All the protein-protein contact areas were calculated using Gromacs tools.

RMSD of an RND1 or RhoD as an indicator of its position relative to the plexin
The RMSD calculation was carried out by first aligning the system by the Cα atoms of the GAP domain of the plexin protomer to which the GTPases is bound to, and then the RMSD was calculated using the Cα atoms of the GTPase with respect to their initial positions in the aligned simulation system.

RMSD of RND1 or RhoD as an indicator of its position relative to the RBD domain
The RMSD calculation was carried out by first aligning the system by the Cα atoms of the RBD domain of the plexin protomer to which the GTPases is bound to, and then the RMSD was calculated using the Cα atoms of the GTPase with respect to their initial positions in the aligned simulation system.

RMSD of RBD as an indicator of its position relative to the corresponding GAP domain
The RMSD calculation was carried out by first aligning the system by the Cα atoms of the GAP domain of the same plexin protomer, and then the RMSD was calculated using the Cα atoms of the RBD with respect to their initial positions in the aligned simulation system.

RMSD of the dimerization helix as an indicator of the stability of the dimer interface
The dimerization helices were first aligned using their Cα atoms, and then the RMSD was calculated using the Cα atoms with respect to their initial position.

The metric for protein-membrane interaction
For each residue of the protein in each simulation snapshot, the number of any lipid molecules within 5 Å of any atom of the residue is calculated. This number was averaged over each simulation (with the first 0.3 μs of the simulation ignored) for each protein residue as a metric for the residue's membrane interaction.

Sequence Alignment
The sequences of human RND1 and RhoD were downloaded from NCBI website. The sequence alignment of the GTPases of the Rho family was performed using the UniProt website (https://www.uniprot.org/).

Protein expression and purification
The coding regions of the intracellular region of mouse plexin B2 with the juxtamembrane region removed (residues 1274-1842) was cloned into a modified pET-28(a) vector (Novagen) that encodes an N-terminal His6-tag followed by a recognition site for human rhinovirus 3C protease.
ArcticExpress (DE3) carrying the expression plasmid was cultured at 37 °C in 100~120 mL LB medium in the presence of Gentamycin overnight. Bacterial cells were scaled up at 30 °C to reach The coding regions of human RhoD (residues 8-194) with the Q75L mutation, which renders the protein catalitically dead and therefore does not hydrolysize GTP, was cloned into the abovementioned modified pET-28(a) vector. The plasmid was transformed into the bacterial strain BL21 (DE3). Protein expression was induced by 0.2 mM IPTG at 16 °C overnight. The protein purification procedure was similar to that for plexin B2, except that all the buffers contained 2 mM MgCl2. The RhoD protein with the Hist6-tag removed was subjected to the final purification step with a Superdex 75 GL 10/30 column with Buffer D containing 20 mM Tris (pH 8.0), 250 mM NaCl, 10 % glycerol (v/v), 2 mM MgCl2, and 2 mM DTT.
To load the protein with GMP-PNP (guanosine 5′-[β,γ-imido]triphosphate) for crystallization, the purified RhoD protein was incubated with GMP-PNP at 20-fold molar ratio to the protein in the exchange buffer containing 20 mM Tris (pH 8.0), 250 mM NaCl, 5 % glycerol (v/v), 7.5 mM EDTA, and 1 mM DTT at RT for 2 hours. After the incubation, 20 mM MgCl2 was added to stop exchange reaction. The protein was then subjected to gel filtration chromatography on a Superdex 75 GL 10/30 column equilibrated with Buffer D to remove excess GMP-PNP.

Crystallization, X-ray data collection and structure determination
Plexin B2 and GMP-PNP-loaded RhoD were mixed at 1:1 molar ratio in a buffer containing 10      No. of Nagatively-charged residues A B the membrane interface regions of Rho GTPases Charges at the membrane interface regions and the C tail Figure 6