The FASEB Journal • Research Communication

Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel b4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents Xingfu Xu,* Fangxiong Zhang,† Gerald W. Zamponi,† and William A. Horne*,1 *Department of Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, USA; and †Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada Rem, Rad, Kir/Gem (RGK) proteins, including Rem2, mediate profound inhibition of high-voltage activated Ca2+ channels containing intracellular regulatory b subunits. All RGK proteins bind to voltage-gated Ca2+ channel b subunit (Cavb) subunits in vitro, but the necessity of the interaction for current inhibition remains controversial. This study applies NMR and calorimetric techniques to map the binding site for Rem2 on human Cavb4a and measure its binding affinity. Our experiments revealed 2 binding surfaces on the b4 guanylate kinase domain contributing to a 156 6 18 mM Kd interaction: a hydrophobic pocket lined by 4 critical residues (L173, N261, H262, and V303), mutation of any of which completely disrupted binding, and a nearby surface containing 3 residues (D206, L209, and D258) that when individually mutated decreased affinity. Voltage-gated Ca2+ channel a1A subunit (Cav2.1) Ca2+ currents were completely inhibited by Rem2 when coexpressed with wild-type Cavb4a, but were unaffected by Rem2 when coexpressed with a Cavb4a site 1 (L173A/ V303A) or site 2 (D258A) mutant. These results provide direct evidence for a low-affinity Rem2/Cavb4 interaction and show definitively that the interaction is required for Cav2.1 inhibition.—Xu, X., Zhang, F., Zamponi, G. W., Horne, W. A. Solution NMR and calorimetric analysis of Rem2 binding to the Ca2+ channel b4 subunit: a low affinity interaction is required for inhibition of Cav2.1 Ca2+ currents. FASEB J. 29, 1794–1804 (2015). www.fasebj.org ABSTRACT

Key Words: RGK proteins • brain • electrophysiology • ion channel • synaptic transmission CA2+ CHANNEL b SUBUNITS (Cavb1–Cavb4) are vital intracellular regulatory proteins that bind with nanomolar affinity to a diverse family of high-voltage activated (HVA) Ca2+ channels (1). These channels are found in the plasma membrane of excitable cells and are composed principally Abbreviations: AID, a1 subunit interaction domain; Cav2.1, voltage-gated Ca2+ channel a1A subunit; Cavb, voltage-gated Ca2+ channel b subunit; CSP, chemical shift perturbation; GK, guanylate kinase; HSQC, heteronuclear single quantum coherence spectroscopy; HVA, high-voltage activated; ITC, isothermal titration calorimetry; MAGUK, membrane-associated

(continued on next page)

1794

of a pore-forming a1 subunit (Cav1.1–Cav1.4; Cav2.1– Cav2.3) and an a2d subunit (a2d12a2d4) that is largely extracellular (2). Among their many regulatory functions, Ca2+ channel b subunits mediate potent RGK protein (Rem, Rem2, Rad, Gem/Kir) inhibition of HVA Ca2+ channels in muscle, neuronal, and endocrine cells (3–5). The molecular details of the Cavb subunit-mediated RGK effects are incompletely understood, in large part because RGK inhibitory mechanisms are complex and appear to vary with RGK and Ca2+ channel a1 subunit subtypes (6–8). It has been shown that all RGK proteins can bind to all Cavb subunits in vitro, but in some cases, direct binding to a Cavb subunit is not required for RGK Ca2+ current inhibition. For instance, 1 study has shown that Gem inhibition of Cav2.1 requires a Cavb3 subunit but that mutations that disrupt Cavb3/Gem binding do not prevent Ca2+ channel inhibition (9). A model is proposed to suggest that Cavb subunit binding to Cav2.1 induces an inhibitory site on Cav2.1 that serves as a target for the C terminus of Gem. This was supported by a further study showing that in the presence of a Cavb subunit, a Gem Cterminal 12-amino-acid peptide could, on its own, inhibit Cav2.1 currents (10). By contrast, Cav1.2 channels, combined with Cavb2a subunits rendered incapable of binding RGK proteins, were still inhibited by Rem and Rad, but not by Gem and Rem2 (6). Adding to this mechanistic diversity, there appears to be multiple ways in which a single RGK protein can inhibit a Ca2+ channel. Three non– mutually exclusive modes of channel inhibition have been identified to date: down-regulation of the number (N) of channels expressed in the plasma membrane by reduced trafficking (11) and accelerated dynamin-dependent endocytosis (6); partial immobilization of a1 subunit voltage sensors resulting in reduced gating current and Qmax (12); and direct inhibition of Ca2+ channel gating (stabilization of low PO gating mode) with no effect on the number of channels trafficked to the membrane (6, 7, 13, 14). 1

Correspondence: Department of Clinical Sciences, 736 Wilson Rd., East Lansing, MI 48824. E-mail: hornewil@cvm. msu.edu doi: 10.1096/fj.14-264499 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

0892-6638/15/0029-1794 © FASEB

Not all RGK inhibitory modes are Cavb subunit dependent. For example, Rem coexpressed with Cav1.2 has been shown to require Cavb2a for down-regulation of N and a decrease in Po gating, but not for a reduction in Qmax (6). Also, not all a1 subunits respond to RGKs in the same way. Rem, for instance, does not affect Cav1.1 charge movement as it does with Cav1.2, whereas Rad does so in a manner that requires the Rad N terminus (7). By contrast, Rem2 inhibits Cav1.2, Cav1.3, and Cav2.2 by reducing PO gating (7, 13). Moreover, Rem2 appears to be unique among the RGKs in that it also has direct effects on Ca2+ channel gating kinetics (8). Clearly, much more experimental work is required to fully understand the molecular details of the functional diversity of RGK inhibition of voltage-gated Ca2+ channel currents. Our study uses techniques in solution NMR spectroscopy and isothermal titration calorimetry (ITC) to examine the structural details of Rem2 binding to the Ca2+ channel Cavb4 subunit. All Cavb subunits are members of the membrane-associated guanylate kinase (MAGUK) protein family that contain a core structure composed of Src homology (SH3) and guanylate kinase (GK) domains (1). Previous studies using site-directed mutagenesis and in vitro binding assays localized the Rem binding site to an ;130 region of the Cavb subunit GK domain that was distinct from the a1 subunit a1 subunit interaction domain (AID) peptide binding site (15). This was replicated in a modeling/mutagenesis study that more precisely localized the Gem binding region to a pocket in the GK domain that is composed of remnant structural motifs that formed the GMP binding site in ancestral guanylate kinase (16). Both studies provided structural information to support the notion that RGK binding does not interfere with Cavb subunit binding to Cav1.2 and vice versa. The modeling/mutagenesis study identified 3 aspartate residues (D194, D270, and D272) and a hydrophobic pocket on the b3 subunit GK domain that support RGK binding (16). Our results confirm the general location of the GK binding site identified in previous studies and provide added detail regarding the amino acids that are affected by the interaction. Mutating b4 D258 (b3 D272 equivalent) to alanine lowers RGK binding affinity and also affects the ability of Rem2 to inhibit Cav2.1 Ca2+ channel currents. Mutating residues in the hydrophobic pocket, b4 L173 and V303 (b3 L187 and V317 equivalents), to alanines completely eliminates Rem2 binding, as well as the ability of Rem2 to inhibit CaV2.1 Ca2+ channel currents. Further mutagenesis reveals a surface for Rem2 binding to Cavb4 that is somewhat different from that previously reported for Rem binding to Cavb3 (16). This may partially explain why Rem2 has unique Ca2+ channel inhibitory properties relative to its RGK relatives. MATERIALS AND METHODS DNA subcloning and mutagenesis The cDNAs encoding the SH3 domain of the Ca2+ channel b4a subunit (b4a38–133, residue numbers 38–133 of the full-length

(continued from previous page) guanylate kinase; N, number of channels expressed at the cell surface; Ni-NTA, nickel-nitroacetic acid; Po, channel open probability; RGK, Rem, Rad, Kir/Gem proteins; SH2, Src homology; TROSY, transverse relaxation-optimized spectroscopy

REM2/b4 INTERACTION REQUIRED FOR CAV2.1 BLOCK

sequence) (17) and the G-domain of the human Rem2 protein (residue numbers 110–286 of the full-length sequence) (18) were subcloned into pET15b (Novagen, Madison, WI, USA) using NdeI and XhoI restriction sites. A cDNA encoding the GK domain of the human Ca2+ channel b4a subunit (residue numbers 167–374 of the full-length sequence) (17) was subcloned into pET-15b using NcoI and XhoI restriction sites to bypass the N-terminal 6His residues. Six histidine residues were incorporated into the C terminus of the GK construct to enable purification by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Site-directed mutagenesis was conducted with the Quickchange (Stratagene, La Jolla, CA, USA) mutagenesis kit according to the manufacturer’s instructions. All constructs were verified by DNA sequencing.

Protein expression and purification Isotopic labeling and purification of a conjoined b4 SH3-GK core protein (329 amino acids; contains SH3 residues 18–134 and GK residues 166–374 of the full-length b4a sequence bridged by 2 serines) was performed according to protocols previously described (19), which contains resonance assignments for SH3-GK fusion protein residues 12–350. [All residue numbers in the manuscript refer to the fusion construct sequence (19). Add 22 or 14 to obtain full-length b4a (17) or b3 (16) GK domain residue numbers, respectively.] To create a SH3-GK core protein in which the GK domain was specifically labeled, the Cavb438-133 SH3 (fusion protein amino acids 44–139) and Cavb4167-374 GK (fusion protein residues 145–352) proteins were first expressed separately in the Escherichia coli strain BL21 Rosetta (DE3) pLysS (EMD, Millipore, Billerica, MA, USA). Subsequently, the SH3 protein was grown in isotope-free Luria broth (LB) medium, and the GK protein was grown in M9 minimal medium containing 15NH4Cl (1 g/L) and U-2H-13C-glucose (2 g/L) (Cambridge Isotope Laboratories, Andover, MA, USA). After sonication and centrifugation, the SH3 protein was purified from the supernatant by Ni-NTA affinity chromatography. Next, the protein was subjected to thrombin cleavage to remove the 6His tag and further purified by FPLC size exclusion chromatography using a Sephacryl 200 column (AKTA; GE Healthcare, Pittsburgh, PA, USA). For purification of the GK domain, a single colony of freshly transformed E. coli harboring pet15b-Cavb4167-374 was inoculated into 20 ml LB medium in 80% (v/v) D2O containing 50 mg/ml ampicillin and grown overnight. One milliliter of the overnight culture was further inoculated into 100 ml M9 media supplemented with 15 NH4Cl, 13C glucose, and 80% (v/v) D2O. After 12-hour growth at 37°C, the cells were pelleted by centrifugation at 4000 rpm and transferred into 1 L 99.9% D2O containing M9 media ingredients and 15NH4Cl (1 g/L) and U-2H-13C-glucose (2 g/L). The cells were further grown at 37°C until the OD600 reached 0.5 AU. Isopropyl-b-D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and protein expression was induced at 37°C for 12 h before cell harvest. The perdeuterated 1H,15N,13Clabeled GK protein was purified from inclusion bodies after sonication and centrifugation. The inclusion bodies were dissolved in 6 M guanidium HCl, and the denatured GK protein was purified by Ni-NTA affinity chromatography. The denatured protein was then refolded at 4°C by rapid dilution (20-fold) of a 10 ml aliquot of 100 mM purified GK into a 200 ml solution containing 5 mM purified SH3, 500 mM NaCl, and 50 mM TrisHCl, pH 8.0. After extensive dialysis against 500 mM NaCl and 50 mM Tris-HCl, pH 8.0, to remove the denaturant, the refolded labeled GK protein in complex with SH3 was further purified by Sephacryl 200 size exclusion chromatography. The Rem2110-286 G-domain protein was expressed in the E. coli strain, Rosetta (DE3) pLysS as an N-terminal 6His fusion protein. The soluble lysate was purified by Ni-NTA affinity chromatography followed by thrombin cleavage and Sephacryal S-200 column

1795

size exclusion chromatography. The reagents 50 mM GDP and 5 mM MgCl2 were included in buffers in all purification steps to prevent Rem2 precipitation. A 16-mer peptide (IERELNGYMEWISKAE) corresponding to the I–II loop AID of the a1A voltage-gated Ca2+ channel was custom synthesized to a purity level .95% (Genescript, Piscataway, NJ, USA). The peptide was used for NMR titration experiments without further purification. Protein purity was analyzed on SDS-PAGE gels, and the protein samples were concentrated via centrifugal filtration using 10 kDa exclusion filters for Rem2, SH3, and GK and a 30 kDa exclusion filter for the SH3-GK complex (Amicon Ultra 15; EMD, Millipore). Protein concentrations were determined by the UV absorbance at 280 nm using theoretical extinction coefficients at 280 nm of (M-1 cm-1) 21,645 (Rem2), 27,055 (SH3-GK), 13,980 (SH3), 11,585 (GK), and 6990 (AID peptide).

NMR spectroscopy NMR experiments were recorded at 298 K with a 900 MHz Bruker Avance spectrometer (Billerica, MA, USA) equipped with a triple resonance TCI CryoProbe. The data were processed with NMRPipe (20) and analyzed using Sparky assignment software (21). The amide backbone assignments of the TROSY-HSQC derived from the GK-specific labeled protein SH3-GK complex were largely transferred from the assignment of the 1H-15N TROSY-HSQC of the deuterated SH3-GK core (19). The assignments were confirmed by 1H-15N-13C-TROSY type 3-D experiments: HNCA, HNCOCA, and CBCACONH. The NMR samples contained 0.5 mM protein (unlabeled SH3 protein in complex with the 2H, 15N, and [13C]-labeled GK domain) in 100 mM NaCl, 2 mM DTT, 0.1% NaN3, 10% (v/v) D2O, and 50 mM sodium phosphate buffer, pH 7.0. For AID peptide titrations, stock solutions of 10 mM peptide were prepared in 100 mM NaCl and 50 mM sodium phosphate buffer, pH 7.0. A baseline 1H-15N TROSY-HSQC spectrum was recorded with an initial sample containing 0.3 mM 2H,15N, and 13 C-labeled SH3-GK or 2H,15N, and 13C-specific labeled GK in complex with unlabeled SH3 in 100 mM NaCl, 2 mM DTT, 10% (v/v) D2O, and 50 mM sodium phosphate buffer, pH 7.0. A series of 1H-15N TROSY-HSQC spectra was then recorded when 1 ml aliquots of unlabeled peptide were added to the solution to a final ratio of peptide to protein of 1.5. To analyze the amide backbone assignment of SH3-GK core in complex with the AID peptide, a 3D 1H-15N NOESY-HSQC was recorded. The averaged chemical shift change of 15N and 1H was calculated with the equation qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Ddavg ¼ DdN2 =50 þ DdH2 =2 in which DdN and DdH represent the chemical shift change of the amide nitrogen and proton, respectively. For Rem2 G-domain titrations, all proteins were exchanged into a buffer containing 50 mM NaCl, 5 mM MgCl2, 1 mM GDP, 2 mM DTT, 10% (v/v) D2O, and 25 mM sodium phosphate buffer, pH 7.4. The first HSQC was recorded with a sample containing 0.3 mM 2H, 15N, and 13C-labeled SH3-GK or 2H,15N, and 13C-labeled GK coupled to unlabeled SH3. A series of 1H15 N TROSY-HSQC spectra was recorded for samples when the unlabeled Rem2 protein was added to reach the ratios of 0.2, 0.5, 1.0, and 2.0. ITC ITC measurements were carried out at 293K using a MicroCal VP-ITC microcalorimeter (GE, Nothampton, MA, USA). All

1796

Vol. 29

May 2015

proteins were dialyzed against the same buffer, and all buffers were degassed before experiments. Repeats of each experiment were performed with different batches of protein. For the AID peptide titration, the AID peptide was buffer exchanged into ITC buffer containing 100 mM NaCl and 50 mM sodium phosphate, pH 7.0, using a PD10 column. Multiple injections of 5 ml of 350 mM peptide were titrated into 1.4 ml of 14 mM b4 SH3-GK core protein (or buffer only, for baseline correction) to a stoichiometric ratio of 3.8:1 (n = 3). For the Cavb4 SH3-GK core protein (n = 3) and its mutants (n = 2 for each mutant) interacting with Rem2, the buffer contained 100 mM NaCl, 5 mM MgCl2, 50 mM GDP, and 25 mM sodium phosphate, pH 7.4. (Protein yields for N261A and H262A mutants were quite low compared with other Cavb4 mutants. This mandated extensive concentration of the proteins and a number of additional repeat experiments to generate reliable ITC curves.) Multiple injections of Cavb4 protein in the concentration range of 400–700 mM were titrated into 1.4 ml of 15–30 mM Rem2 G-domain protein. Data were processed and analyzed with MicroCal Origin software, v.7.0 (GE).

Cell culture and transient transfection HEK tsA-201 cells were grown to 80–90% confluence at 37°C (5% CO2) in DMEM medium (Life Technologies, Grand Island, NY, USA), supplemented with 10% (v/v) fetal bovine serum (HyClone; Thermo Scientific, Pittsburgh, PA, USA), 200 U/ml penicillin, and 0.2 mg/ml streptomycin (Life Technologies). Cells were suspended with 0.25% trypsin/EDTA and plated onto glass coverslips in 35 mm culture dishes (Corning, Corning, NY, USA) at 10% confluence, 24 hours before transfection. The cells were transiently transfected with cDNAs encoding rat Cacna1A (GenBank accession number: NP_037050), rat Cacna2d1 (GenBank accession number: NP_037051), human Cacnb4 (GenBank accession number: NP_001005747) (wild-type, L173A/V303A double mutant, D256A mutant, or D258 mutant), mouse Rem2 (GenBank accession number: NP_542764) or empty vector [pcDNA3.1(+), Life Technologies], and pEGFP-N1 (Clontech Laboratories, Mountain View, CA, USA) as a marker at 1 + 1 + 1 + 0.1 + 0.1 mg with 6 ml Turbofect transfection reagent (Thermo Scientific). Cells were transferred to 30°C 6 hours after transfection and stored for 42 hours before recording.

Electrophysiology Cells on a glass coverslip were transferred into an external bath solution of 20 mM BaCl2, 1 mM MgCl2, 40 mM tetraethylammonium chloride, 65 mM CsCl, 10 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid, and 10 mM glucose, pH 7.4. Borosilicate glass pipettes (Sutter Instrument Co., Novato, CA, USA) (2–4 MV) were filled with internal solution containing 140 mM CsCl, 2.5 mM CaCl2, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 2 mM Na-ATP, and 0.3 mM Na-GTP, pH 7.3. Whole-cell patch clamp recordings were performed by using an EPC 10 amplifier (HEKA Elektronik, Bellmore, NY, USA) linked to a personal computer equipped with Pulse (V8.65) software (HEKA Elektronik). After seal formation, the membrane beneath the pipette was ruptured, and the pipette solution was allowed to dialyze into the cell for 1–2 minutes before recording. Currents were elicited by depolarization from a holding potential of 290 mV to various test potentials for 100 ms with an interpulse interval of 1 s. Voltagedependent currents were corrected for leak by using an online P/4 subtraction paradigm. Data were recorded at 10 kHz and filtered at 2.9 kHz. Data analysis was performed by using online analysis built in Pulse software, and graphs were prepared by using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Error bars plotted represent the mean values 6 SE.

The FASEB Journal x www.fasebj.org

XU ET AL.

RESULTS GK domain specific labeling by in vitro corefolding A Cavb4 core domain construct (329 amino acids) composed of the SH3 domain (residues 18–134 of the fulllength sequence) and GK domain (residues 166–374 of the full-length sequence) bridged by 2 serines was expressed, 15 N and 13C labeled, and perdeuterated in E. coli. The removal of a large serine-rich loop (33 amino acids) in the hook region between the SH3 and GK domains resulted in expression of a stable, monomeric protein construct suitable for multidimensional NMR experiments. This is confirmed by the well-dispersed 1H, 15N-TROSY-HSQC spectrum of the fully labeled Cavb4 SH3-GK construct (Fig. 1A). The backbone assignments of this construct were completed with a suite of 3-dimensional experiments and reported previously (19). As can be seen in the figure, there is considerable resonance overlap in the spectrum, especially in the central region. This has hindered the ability to perform chemical shift analysis using this labeled construct to study b4 protein-protein interactions. To address the problem of resonance overlap, a domainspecific labeling protocol was developed for the Cavb4 GK domain. After multiple failed attempts to express a singular GK domain that folded properly, we developed a refolding strategy in which the purified, folded, and unlabeled SH3 domain was used as a chaperone to assist in the folding of labeled GK. When the fifth b-strand (residues 166–171) of the SH3 domain was included on the N terminus of GK, a folded SH3 lacking the fifth strand was capable of assisting with in vitro refolding of GK. This enabled us to 2H,15N,13C label the GK domain specifically while leaving the SH3 unlabeled in the final Cavb4 core construct: (SH3)-2H15N GK. As shown in Fig. 1B, this dramatically improved the spectral resolution of the GK domain 1H-15N TROSY-HSQC spectrum.

able to bind protein we performed a chemical shift perturbation analysis with a Cava peptide known to interact with the GK domain. A 16-mer peptide sequence derived from the Cav2.1 a1 subunit I–II loop was synthesized for these experiments. The peptide lacks 2 N-terminal Gln residues compared with an 18-mer AID peptide used by other groups (22, 23); however, as shown in a previous ITC analysis, these 2 residues were not absolutely required for AID binding to Cavb subunits (24). Our experiments reveal large chemical shift changes in 1H,15N-TROSY-HSQC spectra following titration of the 16-mer peptide into NMR samples containing either the 2H,15N-labeled b4 conjoined SH3-GK core (Fig. 2A) or the core complex composed of the refolded and specifically labeled 2H,15N GK domain bound to SH3 (Fig. 2B). Residues M182, L186, L328, E329, and D330, shown previously to be 5 key residues in the Cavb4 a1 subunit binding pocket (1), are highlighted in both figures as examples of the large chemical shifts that are typical of high-affinity binding. For each titration, only 2 sets of cross-peaks representing free and peptide bound states of Cavb4 were detected, indicating that the interaction is in a slow exchange regime. Nearly all the chemical shift change patterns were maintained in the titrations using the labeled GK domain (Fig. 2B) compared with the titrations using the labeled Cavb4 SH3-GK core (Fig. 2A). This demonstrates the equivalent folding and binding ability of the 2 Cavb4 core constructs. Importantly, the ability to perform chemical shift perturbation analysis was dramatically enhanced when the SH3 resonances were removed by specifically labeling the GK domain. The complete AID peptide perturbation analysis is shown in Fig. 2C. Importantly, residues M182 and L328 are 2 of 5 residues in the b4 binding pocket shown in a previous study (24) to have the greatest impact on binding affinity (.10-fold reduction) when mutated to alanine.

AID peptide chemical shift perturbations

Rem2 G-domain titration into Cavb4 SH3-GK core structures

To determine whether the purified conjoined SH3-GK and SH3 bound GK constructs were properly folded and

To determine whether Rem2 binds to Cavb4, we used the GDP-bound form of the Rem2 G-domain to perform

Figure 1. 2H,15N -specific labeling of the Ca2+ channel b4 subunit GK domain. A) 1H-15N TROSY-HSQC spectra of the conjoined 2 H,15N- labeled Cavb4 SH3-GK core domains. B) 1H-15N TROSY-HSQC spectra of the specifically labeled (SH3)-2H,15N-GK domain bound to unlabeled Cavb4 SH3. Note the substantially reduced resonance overlap in the HSQC of (SH3)-2H,15N GK.

REM2/b4 INTERACTION REQUIRED FOR CAV2.1 BLOCK

1797

Figure 2. NMR chemical shift perturbations invoked by Ca2+ channel a1A AID peptide binding to the b4 subunit SH3-GK core structure. A) Overlay of the 1H-15N TROSY-HSQC spectra of the 2H,15N-labeled conjoined Cavb4 SH3-GK core domains before (black) and after (red) the titration of the a1A AID peptide to the ratio of 1.5:1. Residues L186, M182, L328, E329, and D330, shown previously to be located in the Cavb4 a1 subunit binding pocket, are labeled as examples of the largest chemical shifts (arrows). B) Overlay of the 1H-15N TROSY HSQC spectra of the 2H,15N labeled GK domain of the bound SH3GK core before (black) and after (red) the titration of the peptide to the ratio of 1.5:1. Residues M182, L186, L328, E329, and D330 are again shown as examples of large chemical shifts (arrows). Note that they are identical to the shifts in A. C) Plot of all a1A AID-induced chemical shift changes (red bars) against the residues numbers of the Cavb4 subunit GK domain. Residue numbers correspond to those of the conjoined SH3-GK fusion protein construct described previously (19). M182 and L328 are labeled as the largest chemical shifts. [In this and all subsequent figures, residue numbers refer to the fusion construct sequence (19). Add 22 or 14 to obtain full-length b4a (17) or b3 (16) GK domain residue numbers, respectively.]

a chemical shift perturbation analysis. Both GDP and Mg2+ were included in the buffers throughout the purification process and in all NMR experiments to stabilize the Rem2 G-domain. When the unlabeled Rem2 G-domain was titrated into the perdeuterated, conjoined 2H,15N-labeled Cavb4 SH3-GK core structure, complex formation was evident from general line broadening and disappearance of cross-peaks (Fig. 3A). Some cross-peaks showed very rapid chemical shift changes, as line broadening was evident as early as the third titration point. In contrast to results with the AID peptide, no changes representing population averaged chemical shifts of the bound state were detected for residues in the presence of Rem2. This indicates that the interaction is in an intermediate exchange regime. Fig. 3A clearly shows the difficulty in performing chemical shift perturbation analysis of the central region of the completely labeled SH3-GK core structure that results from severe resonance overlap. Interestingly, however, most of the cross-peaks in the welldispersed region of the spectrum that undergo rapid disappearance can be assigned to the residues of GK domain. This suggests that the Rem2 binding site is 1798

Vol. 29

May 2015

located principally on the GK domain. This was confirmed by performing Rem2 G-domain titration against a core complex that contained the 2H,15N-labeled labeled GK domain bound to an unlabeled SH3 (Fig. 3B). GK domain residues experiencing significant line broadening at the third titration point have been identified as possible points of contact for Rem2 (see below). Comparison of the interface for AID and Rem2 binding to the Cavb4 GK domain To confirm that there is little overlap between the AID peptide and Rem2 binding sites on Cavb4, we created a surface map for each site based on our perturbation analysis data. Residues on the Cavb4 subunit that showed significant NMR chemical shift changes in response to binding of the a1 AID peptide are mapped onto the SH3GK crystal structure in Fig. 4A. A continuous surface was found to surround an AID binding pocket previously identified by X-ray crystallography (1). Some residues such as F189, R193, F194, and L339 showing chemical shifts are

The FASEB Journal x www.fasebj.org

XU ET AL.

Figure 3. NMR evidence of Rem2 G domain binding to the Ca2+ channel b4 subunit GK domain. A) Overlay of 1H-15N TROSYHSQC spectra of the conjoined 2H,15N-labeled Cavb4 SH3-GK core structure before (black) and after (red) adding unlabeled Rem2 G-domain to a ratio 2:1. B) Overlay of 1H-15N TROSY-HSQC spectra of the 2H,15N-labeled GK domain bound to unlabeled SH3 before (black) and after (red) adding unlabeled Rem2 G-domain to 2:1 ratio. The cross-peaks experiencing substantial line broadening on Rem2 addition are labeled with residue assignments (green labels). Residue D256, which is not perturbed by Rem2 but is used in a later electrophysiology experiment, is also labeled (black label).

located relatively far from the binding groove. These chemical shifts are likely caused by small side chain conformational changes of aromatic residues. The Rem2 G domain interaction site was mapped to a large concaved region on the flip side of the GK domain (180° rotation; Fig. 4B) that includes remnants of the nucleotide biding pocket of canonical GK proteins. The binding site also contains residues just proximal to 310 helix sequences 2 and 3 and residues in a helix 6. As seen in

Fig. 5B, the residues are located in 5 small contiguous subdomains: 1 (G170, L173, and G175); 2 (Q183, A185, L186, and F187); 3 (A205, D206, I207, and L209); 4 (A257, D258, I260, N261, H262, Q265, and I267); and 5 (H300, N302, V303, Q304, L305, V306, A307, and A308). These surface representations clearly indicate that the Cavb subunit binding regions for AID and Rem2 are distinct and that a direct competition for binding between the AID and Rem2 is unlikely.

Figure 4. Solution NMR-derived surface representations of binding regions for the a1A AID peptide and Rem2 G-domain on the GK domain of the Ca2+ channel b4 subunit. A) Ca2+ channel a1A AID binding site derived from chemical shift perturbations and mapped onto the GK domain of the Cavb4 subunit SH3-GK crystal structure (Protein Data Bank ID code 1VYV). Residues of the GK domain experiencing chemical shift changes .0.12 ppm on adding the AID peptide are labeled in red; those .0.08 ppm but ,0.12 ppm are labeled in orange. The AID peptide in complex with b4 is shown in ribbon diagram form (blue) with side chains shown as sticks (modeled from Protein Data Bank ID code 1VYT using Modeler 9.10; University of California, San Francisco, CA, USA). B) Rem2 G-domain binding site on the GK domain of the Cavb4 subunit SH3-GK core structure as derived from chemical shift perturbations. Residues showing Rem2 G-domain induced line broadening because of rapid intermediate chemical exchange are labeled in green.

REM2/b4 INTERACTION REQUIRED FOR CAV2.1 BLOCK

1799

Figure 5. Isothermal titration calorimetry analysis of a1A AID peptide and Rem2 G-domain binding to the Cavb4 SH3-GK core protein. A–L) (Upper) Exemplar raw thermograms after baseline correction. (Lower) Data processed with Microcal Origin software (7.0). Solid lines represent best fits to a 1-site binding model. A) a1A AID peptide (350 mM) titrated into Cavb4 SH3-GK core (16 mM). B) Cavb4 SH3-GK core (800 mM) titrated into Rem2 G-domain (45 mM). C–L) The core alanine mutant was titrated into the wild-type Rem2 G-domain. Protein amounts [expressed as SH3GK (mM) and Rem2 (mM)] were as follows: L173A (550, 20); D206A (600, 30); L209A (600, 22); D256A (480, 18); D258A (700, 35); N261A (500, 15); H262A (400, 20); Q265A (600, 33); N302A (600, 21), and V303A (700, 30).

1800

Vol. 29

May 2015

The FASEB Journal x www.fasebj.org

XU ET AL.

Identification of critical GK residues required for weak interaction of Rem2 with the Cavb4 GK domain Rapid signal broadening caused by intermediate exchange prevents an accurate NMR measurement of the binding affinity for the Rem2 G-domain/Cavb4 subunit GK domain interaction. We therefore used ITC for this purpose (Fig. 5). For comparison purposes, we first performed an ITC experiment using the same 16-mer AID peptide used for NMR titrations against the b4 SH3-GHK construct. We observed a thermogram characteristic of a high-affinity, saturable interaction (Fig. 5A). The reaction is exothermic and has a calculated equilibration dissociation constant (Kd) of 341 6 13 nM. This is weaker than a previously published ITC-determined affinity of an 18-mer Cav2.1 peptide binding to Cavb2a (24) and is likely attributed to a charge difference (use of nonacetylated peptide) and the lack of 2 N-terminal Asn residues. For Rem2 binding, experiments were performed by including the Rem2 G-domain in the sample cell and titrating in wild-type or mutant Cavb4 SH3-GK proteins (Fig. 5B–L). The wild-type SH3-GK thermogram reveals a typical weak interaction curve that does not reach saturation even at the final titration point (Fig. 5B). The interaction is endothermic with a calculated Kd of 156 6 18 mM. This affinity falls into the low end of measuring limits of isothermal titration calorimetry (25). Of the 26 Cavb4 GK domain surface residues identified by NMR to be perturbed by the interaction with Rem2 (Fig. 4), we found 9 that had exposed side chains by examining the Cavb4 core crystal structure. We posited that these surface residues were most likely to be involved in direct binding with Rem2. To test this hypothesis, we individually mutated each of the 9 residues to alanine and repeated the ITC experiments. These surface mutations would not be predicted to cause global structural changes given that their side chains are not involved in internal chemical bonds. Thus, changes in binding properties would serve as a good estimate of the contribution of each residue to binding affinity. The results of these experiments were as follows: thermograms of Q265A and N302A were not different than wild type, indicating that these residues are not directly involved in Rem2 binding; thermograms for L173A, N261A, H262A, and V303A (Figs. 5C, H, I, and L,

respectively) were essentially flat (after correcting for the heat of dilution), indicating that these residues are essential for binding of Rem2; and thermograms for D206A, L209A, and D258A (Fig. 5D, E, and G, respectively) showed some evidence of very low-affinity binding, although too low to calculate an accurate Kd. This indicates that these residues are essential but may not be absolutely required for binding. Thus, the results of our alanine-scanning mutagenesis experiments led us to identify a Rem2 binding pocket formed by L173, V303, V306, N261, and H262 and a contiguous binding surface formed by D206, L209, and D258 (Figs. 6 and 7). As an additional experiment, we mutated D256 to alanine because, although not perturbed in our chemical shift analysis (Fig. 3B), it has previously been identified as important for the Kir/Gem interaction with Cavb3 (16). Consistent with our NMR data, the D256A mutation had no effect on Rem2 binding to Cavb4 (Fig. 5F). To further validate our binding results, a double mutant, Cavb4 SH3-GK-L173A/V303A, was expressed and 2H-15Nlabeled, and used in Rem2 chemical shift perturbation analysis experiments. As seen in the 1H,15N-TROSY-HSQC overlay (Supplemental Fig. S1), small chemical shift changes were observed that were likely attributable to the 2 mutated residues. The fact that the mutations had no detectable effect on the overall dispersion of the remaining resonances indicates that they had minimal effects on protein folding. To determine whether the double mutation completely eliminated Rem2 G domain binding, the unlabeled G-domain was titrated into 2H,15N-labeled Cavb4a SH3-GKL173A/V303A. As shown in Supplemental Fig. S2, neither chemical shift nor line broadening of NMR cross-peaks was observed, indicating that these 2 mutations completely abolished the interaction of the Rem2 G-domain with the Cavb4 SH3-GK core domain. Functional effects of mutating Cavb4 residues required for Rem2 binding To assess the functional importance of residues L173 and V303, the mutations were introduced into an expression construct for Cavb4a. This construct (or wild-type Cavb4a) was then coexpressed with Cav2.1 and Cava2d in tsA-201

Figure 6. Ca2+ channel b4 subunit GK domain binding site for Rem2 G-domain identified by NMR-directed alanine scanning mutagenesis and isothermal titration calorimetry. Residues for which alanine substitutions eliminated Rem2 G domain binding, L173, V303, N261, and H262, are shown as red spheres. Residues for which individual substitutions resulted in decreased affinities, D206, L209, and D258, are shown as orange spheres. Residues for which substitutions had no effect on binding, N302 and Q265, are shown as blue spheres.

REM2/b4 INTERACTION REQUIRED FOR CAV2.1 BLOCK

1801

Figure 7. Mesh surface diagram of the Ca2+ channel b4 subunit-Rem2 G domain binding site. NMR perturbation analysis reveals that the site is composed of 1) a primary pocket formed by 1 residue on the loop between b-strand 6 and a-helix 3 (L173), 2 residues on the very distal portion of the loop between b-strand 8 and 310-helix 3 (N261 and H262), and 2 residues on the distal portion of the a-helix 6 (V303 and V306); and 2) a supporting surface formed by 2 residues from the loop between b-strand 5 and a-helix 4 (D206 and D209) and 1 residue from b-strand 6 (D258). Residue numbers shown are based on the CavSH3-GK core construct in ref. 19 and correspond to L195, D228, L231, D280, N283, H284, V325, and V328 of the full-length Cavb4a sequence. The mesh diagram was created in PyMOL (v. 1.5) using the complete structure of the Cavb4 GK domain. The a-helices, b-strands, and loops are hidden to highlight the binding pocket. Amino acid residues are shown as sticks.

cells in the presence and absence of a cotransfected Rem2 construct. In accordance with what we had observed previously with Cav2.2 + Cavb3 channels (26), coexpression of Rem2 in cells expressing Cav2.1 channels + wild-type Cavb4 shows that whole cell currents were virtually abolished in the presence of Rem2 (Fig. 8). By contrast, in cells expressing the L173/V303 double mutant, current densities were unaltered by Rem2, in agreement with the notion that this mutant was unable to interact with Rem2. The double mutant did not significantly alter current densities or voltage-dependent gating compared with the wild-type Cavb4a subunit, indicating that the physical association of the Cb4a subunit with Cav2.1 is not affected by the mutation. We also tested 2 additional Cavb4a mutants (D256A and D258A) on Cav2.1 channel function. In the absence of Rem2, both of these mutants behaved like wild-type Cavb4a with regard to current density. In the presence of the D256A mutant, Cav2.1 current densities were significantly reduced, whereas in the case of the D258A mutant, Rem2 had little effect. These data fit with the observation that strength of Rem2 binding affinity for Cavb4 is important for Rem2 inhibition of Cav2.1 Ca2+ currents. DISCUSSION NMR and functional assays to study weak protein-protein interactions Weak (Kd $ 1024) protein-protein interactions are poorly understood and yet extremely important for many 1802

Vol. 29

May 2015

cellular events, including rapid and transient assembly of regulatory protein complexes and signal transduction turnover (27). Such events play key roles in fine-tuning synaptic vesicle release at nerve terminals (2). The reason for our incomplete understanding of weak proteinprotein interactions lies in the technical difficulty in characterizing them. They do not typically lend themselves to study by conventional biochemical methods, including X-ray crystallography. This barrier can be overcome by using techniques in NMR spectroscopy, especially when combined with highly sensitive functional assays (28); however, this approach is not without its limitations. In particular, the complexity of NMR spectra increases with protein size, often resulting in difficult to discern resonance overlaps in all dimensions of a 3D NMR experiment. We studied the low-affinity interaction between the 37 kDa SH3-GK MAGUK core of the Ca2+ channel b4 subunit and the 22 kDa G-domain of the RGK protein, Rem2, by combining NMR chemical shift perturbation (CSP) analysis and ITC with site-directed mutagenesis to map and characterize the Rem2/Cavb4 subunit interaction. At 37 kDa, resonance overlap was significant for the labeled SH3-GK protein and thus pushed the CSP analysis technique beyond its size limitation. We overcame this by purifying the SH3 domain separately and using it as an in vitro chaperone to fold the specifically labeled GK domain (which could not be folded on its own). This enabled nearly complete assignment of GK domain resonances and opened the way for clear CSP analysis. (It will be interesting to see whether this technique has wide applications in the field of MAGUK protein structure and function.) Thus, coupled with the patch-clamp electrophysiology technique, NMR and ITC have permitted us to show directly, and for the first time, that a weak proteinprotein interaction is absolutely essential for potent Rem2 inhibition of Cav2.1 Ca2+ channel currents. Comparison to other RGK/b subunit interaction studies This study has provided the first NMR evidence for a direct low-affinity interaction of an RGK protein with a binding site on the voltage-gated Ca2+ channel b subunit. Interestingly, the surface map of the Rem2 binding site on Cavb4 generated from our experiments (Fig. 7) differs somewhat from that determined by a mutagenesis/ molecular modeling study of the interaction of Kir/Gem with Cavb3 (16). Although both studies identify the hydrophobic pocket that is bordered by L173 and N261 (L187 and N275 in Cavb3) as being critical for binding of Kir/Gem and Rem2, NMR analysis of Rem2 binding to Cavb4 indicates that the negatively charged surface formed by D180, D256, and D258 (D194, D270, and D272 in Cavb3) and important for Kir/Gem binding is not required for binding of Rem2 (D180 and D256 charges in particular). By contrast, the surface formed by L209, D206, and D258 in Cavb4 contributes to binding of Rem2. Individual alanine substitutions of these residues reduce affinity but do not eliminate binding. Interestingly however, the reduced affinity caused by D258A was enough to eliminate Rem2’s ability to inhibit

The FASEB Journal x www.fasebj.org

XU ET AL.

Figure 8. Rem2-induced Cav2.1 current inhibition is reversed by certain Cavb4a mutants. Average current-voltage relationships for currents recorded in cells expressing Cav2.1 with or without coexpressing Rem2. For wild-type Cavb4a, Cav2.1 current densities were significantly smaller in Rem2 cotransfected cells than those in cells not expressing Rem2 (P , 0.0001 at 25 mV). For double mutant Cavb4a (L173A/ V303A), however, Cav2.1 current densities were similar between Rem2 coexpressing cells and cells not expressing Rem2 (P = 0.7061 at 25 mV). The double mutations in Cavb4a per se did not affect the overall Cav2.1 current densities relative to those seen with wild-type Cavb4a (P = 0.6198 at 25 mV). The D256A mutation did not affect the ability of Rem2 to reduce current densities (P = 0.003 at 15 mV), whereas D258A shows similar current densities in the presence and absence of Rem2 (P = 0.5151 at 25 mV). Ten experiments per condition are included in the current-voltage data.

Cav2.1 Ca2+ currents in HEK cells (Fig. 8). This result supports the finding of previous laboratories showing that Cavb constructs containing the D258A mutation in a triple mutant construct were unable to support Ca2+ current inhibition by Rem, Rem2, or Gem (6, 9, 16). Our results may explain why B´eguin et al. concluded from their studies that there may be subtle differences in the way Rem2 associates with Cavb3 compared with Rad and Rem (16); however, whether this explains the result that a Gem/b3 subunit interaction is not required for Gem inhibition of Cav2.1 (9) remains to be determined. Our contrasting results raise the interesting possibility that there is a yet to be fully characterized structural specificity underlying RGK inhibition of Ca2+ channels. The results also suggest that future studies examining the role of b4-Rem2 interactions in physiologic processes would benefit from using the b4 L173/V303 double mutant to ensure that Rem2 binding has been eliminated. Diversity of RGK protein/b subunit interactions Sequence comparisons of RGK proteins from a number of genetically diverse organisms suggest that their ability to modulate Ca2+ channels arose from an ancestor that predates the protostome and deuterostome split that occurred .500 million years ago (29). Key RGK Gdomain loop residues important for binding to Ca2+ channel b subunits are highly conserved throughout evolution (R196, V/L223, and H225), arguing that the RGK-b subunit interaction is very important to the function of the RGK protein class. Ca2+ channel b subunit GK residues, including residues of the RGK binding site, are also highly conserved (30); however, this is a general feature of the entire GK domain, and sequence comparisons do not point to any one region as a potential binding site. Our results add to the evidence that the Rem2 site has evolved as a distinct site from that of the ancestral GMP REM2/b4 INTERACTION REQUIRED FOR CAV2.1 BLOCK

binding site of guanylate kinase. There is some structural overlap between this site and the location of Walker A and B sequences in P-loop kinases (b-strand 6 and b-strand 8 and proximal loop sequence between b-strands 8 and 9, respectively); however, the Cavb4 sequence lacks key residues necessary for binding the a-phosphate of GMP. Moreover, although b-strands 2 and 4 (equivalents of b-strands 7 and 9 in the b4 subunit, respectively) are key structures involved in GMP binding, they do not play a role in the binding of Rem2. Identifying this as a distinct site is consistent with the notion that MAGUKs have evolved from GKs by accelerated divergence (31). The physiologic importance of the RGK-Cavb subunit interactions remains to be fully elucidated; however, preliminary studies have shown that cellular responses are both cell type and subcompartment specific. In the case of Rem2, direct inhibition of nerve terminal Cav2 channels would be expected to cause immediate inhibition of synaptic vesicle release (13), whereas negative feedback inhibition of cell soma Cav1 channels would have longer-term effects on activity-dependent Rem2 signaling pathways (18, 32, 33). As Rem2, Cavb4, and Cav2.1 are all localized to neurons, and most excitatory CNS synapses contain Cav2.1, our results should contribute in a substantive way to the understanding of the role that neuronal voltage-gated Ca2+ channels play in health and disease. The authors thank Dr. Li Li and Ms. Heather De FeijterRupp for excellent technical support in all aspects of the project. This work was supported in part by U.S. National Institutes of Health, National Institute of Neurological Disorders and Stroke Grant NS42600 (to W.A.H.), and National Science Foundation Grant IOS 0719242 (to W.A.H.). F.Z. is supported by a postdoctoral fellowship from Alberta Innovates–Health Solutions. G.W.Z. is supported by grants from the Canadian Institutes for Health Research and from the Natural Sciences and Engineering Research Council and holds a Canada Research Chair. The authors declare no conflicts of interest.

1803

REFERENCES 1. Buraei, Z., and Yang, J. (2010) The b subunit of voltage-gated Ca2+ channels. Physiol. Rev. 90, 1461–1506 2. Simms, B. A., and Zamponi, G. W. (2014) Neuronal voltage-gated calcium channels: structure, function, and dysfunction. Neuron 82, 24–45 3. Correll, R. N., Pang, C., Niedowicz, D. M., Finlin, B. S., and Andres, D. A. (2008) The RGK family of GTP-binding proteins: regulators of voltage-dependent calcium channels and cytoskeleton remodeling. Cell. Signal. 20, 292–300 4. Flynn, R., and Zamponi, G. W. (2010) Regulation of calcium channels by RGK proteins. Channels (Austin) 4, 434–439 5. Yang, T., and Colecraft, H. M. (2013) Regulation of voltagedependent calcium channels by RGK proteins. Biochim. Biophys. Acta 1828, 1644–1654 6. Yang, T., Puckerin, A., and Colecraft, H. M. (2012) Distinct RGK GTPases differentially use a1- and auxiliary b-binding-dependent mechanisms to inhibit Cav1.2/Cav2.2 channels. PLoS ONE 7, e37079 7. Beqollari, D., Romberg, C. F., Meza, U., Papadopoulos, S., and Bannister, R. A. (2014) Differential effects of RGK proteins on L-type channel function in adult mouse skeletal muscle. Biophys. J. 106, 1950–1957 8. Seu, L., and Pitt, G. S. (2006) Dose-dependent and isoformspecific modulation of Ca2+ channels by RGK GTPases. J. Gen. Physiol. 128, 605–613 9. Fan, M., Buraei, Z., Luo, H. R., Levenson-Palmer, R., and Yang, J. (2010) Direct inhibition of P/Q-type voltage-gated Ca2+ channels by Gem does not require a direct Gem/Cavbeta interaction. Proc. Natl. Acad. Sci. USA 107, 14887–14892 10. Fan, M., Zhang, W. K., Buraei, Z., and Yang, J. (2012) Molecular determinants of Gem protein inhibition of P/Q-type Ca2+ channels. J. Biol. Chem. 287, 22749–22758 11. B´eguin, P., Nagashima, K., Gonoi, T., Shibasaki, T., Takahashi, K., Kashima, Y., Ozaki, N., Geering, K., Iwanaga, T., and Seino, S. (2001) Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem. Nature 411, 701–706 12. Yang, T., Xu, X., Kernan, T., Wu, V., and Colecraft, H. M. (2010) Rem, a member of the RGK GTPases, inhibits recombinant Cav1.2 channels using multiple mechanisms that require distinct conformations of the GTPase. J. Physiol. 588, 1665–1681 13. Chen, H., Puhl III, H. L., Niu, S. L., Mitchell, D. C., and Ikeda, S. R. (2005) Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density. J. Neurosci. 25, 9762–9772 14. Finlin, B. S., Mosley, A. L., Crump, S. M., Correll, R. N., Ozcan, S., Satin, J., and Andres, D. A. (2005) Regulation of L-type Ca2+ channel activity and insulin secretion by the Rem2 GTPase. J. Biol. Chem. 280, 41864–41871 15. Finlin, B. S., Correll, R. N., Pang, C., Crump, S. M., Satin, J., and Andres, D. A. (2006) Analysis of the complex between Ca2+ channel b-subunit and the Rem GTPase. J. Biol. Chem. 281, 23557–23566 16. B´eguin, P., Ng, Y. J., Krause, C., Mahalakshmi, R. N., Ng, M. Y., and Hunziker, W. (2007) RGK small GTP-binding proteins interact with the nucleotide kinase domain of Ca2+-channel b-subunits via an uncommon effector binding domain. J. Biol. Chem. 282, 11509–11520

1804

Vol. 29

May 2015

17. Helton, T. D., and Horne, W. A. (2002) Alternative splicing of the b4 subunit has a1 subunit subtype-specific effects on Ca2+ channel gating. J. Neurosci. 22, 1573–1582 18. Finlin, B. S., Shao, H., Kadono-Okuda, K., Guo, N., and Andres, D. A. (2000) Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases. Biochem. J. 347, 223–231 19. Xu, X., and Horne, W. A. (2014) ¹H, ¹³C, and ¹5N backbone resonance assignments of the 37 kDa voltage-gated Ca²⁺ channel b4 subunit core SH3-GK domains. Biomol. NMR Assign. 8, 217–220 20. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 21. Goddard, T.D., Kneller, D.G., SPARKY 3, University of California, San Francisco. 22. Opatowsky, Y., Chen, C. C., Campbell, K. P., and Hirsch, J. A. (2004) Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain. Neuron 42, 387–399 23. Van Petegem, F., Clark, K. A., Chatelain, F. C., and Minor, D. L., Jr. (2004) Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain. Nature 429, 671–675 24. Van Petegem, F., Duderstadt, K. E., Clark, K. A., Wang, M., and Minor, D. L., Jr. (2008) Alanine-scanning mutagenesis defines a conserved energetic hotspot in the Cavalpha1 AID-Cavbeta interaction site that is critical for channel modulation. Structure 16, 280–294 25. Rajarathnam, K., and R¨osgen, J. (2014) Isothermal titration calorimetry of membrane proteins - progress and challenges. Biochim. Biophys. Acta 1838(1 Pt A), 69–77 26. Flynn, R., Chen, L., Hameed, S., Spafford, J. D., and Zamponi, G. W. (2008) Molecular determinants of Rem2 regulation of N-type calcium channels. Biochem. Biophys. Res. Commun. 368, 827–831 27. Nooren, I. M., and Thornton, J. M. (2003) Diversity of proteinprotein interactions. EMBO J. 22, 3486–3492 28. Vaynberg, J., and Qin, J. (2006) Weak protein-protein interactions as probed by NMR spectroscopy. Trends Biotechnol. 24, 22–27 29. Puhl III, H. L., Lu, V. B., Won, Y. J., Sasson, Y., Hirsch, J. A., Ono, F., and Ikeda, S. R. (2014) Ancient origins of RGK protein function: modulation of voltage-gated calcium channels preceded the protostome and deuterostome split. PLoS ONE 9, e100694 30. Ebert, A. M., McAnelly, C. A., Handschy, A. V., Mueller, R. L., Horne, W. A., and Garrity, D. M. (2008) Genomic organization, expression, and phylogenetic analysis of Ca2+ channel b4 genes in 13 vertebrate species. Physiol. Genomics 35, 133–144 31. Leipe, D. D., Koonin, E. V., and Aravind, L. (2003) Evolution and classification of P-loop kinases and related proteins. J. Mol. Biol. 333, 781–815 32. Flynn, R., Labrie-Dion, E., Bernier, N., Colicos, M. A., De Koninck, P., and Zamponi, G. W. (2012) Activity-dependent subcellular cotrafficking of the small GTPase Rem2 and Ca2+/CaMdependent protein kinase IIa. PLoS ONE 7, e41185 33. Ghiretti, A. E., Moore, A. R., Brenner, R. G., Chen, L.-F., West, A. E., Lau, N. C., Van Hooser, S. D., and Paradis, S. (2014) Rem2 is an activity-dependent negative regulator of dendritic complexity in vivo. J. Neurosci. 34, 392–407

The FASEB Journal x www.fasebj.org

Received for publication September 30, 2014. Accepted for publication December 22, 2014.

XU ET AL.