Structural basis for rare earth element recognition by

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Structural basis for rare earth element recognition by Methylobacterium extorquens lanmodulin Erik C. Cook, Emily R. Featherston, Scott Anthony Showalter, and Joseph A. Cotruvo, Jr. Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01019 • Publication Date (Web): 23 Oct 2018 Downloaded from http://pubs.acs.org on October 25, 2018

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Structural basis for rare earth element recognition by Methylobacterium extorquens lanmodulin Erik C. Cook,1 Emily R. Featherston,1 Scott A. Showalter,1,2,* and Joseph A. Cotruvo, Jr.1,* 1

Department of Chemistry, The Pennsylvania State University, University Park, PA 16802

2

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802 Abstract: Lanmodulin (LanM) is a high-affinity lanthanide (Ln) binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with CaII binding, but it undergoes a metal-dependent conformational change with 100-million-fold selectivity for LnIIIs and YIII over CaII. Here we present the NMR solution structure of LanM complexed with YIII. This structure reveals that LanM features an unusual fusion of adjacent EFhands, resulting in a compact fold to our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein’s picomolar affinity for LnIIIs, and it suggests a role of unusual Ni+1–H•••Ni hydrogen bonds, in which LanM’s unique EF-hand proline residues are engaged, in selective LnIII recognition. This work sets the stage for a detailed mechanistic understanding of LanM’s Ln-selectivity, which may inspire new strategies to bind, detect, and sequester these technologically important metals. The similar coordination preferences and ionic radii of lanthanides (LnIIIs) and CaII have long been exploited in biochemical research to probe calcium binding sites in proteins, such as the ubiquitous eukaryotic CaII sensor, calmodulin (CaM).1-3 The metal recognition motifs in these proteins are EF-hands, ~29-residue helix-loop-helix motifs typically comprising a 12-amino-acid, carboxylate-rich metal-binding loop flanked by entering and exiting α-helices.4, 5 These sequences, often found in pairs, are responsive to CaII concentrations in the high nanomolar to millimolar range, depending on the protein.4 CaM, for example, possesses two pairs of EF-hands (EF1/2 and EF3/4) connected by a flexible helical linker,6, 7 and metal ion binding triggers conformational changes to mediate interaction with various substrate proteins.8 EF-hands have also been engineered into “lanthanide-binding tags” (LBTs), short peptides with higher selectivity for LnIIIs, which take advantage of TbIII or EuIII luminescence for labelling applications.9, 10 Only recently, however, have Lns also been established to play essential roles in biology, in the catalytic sites of certain pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases (ADHs), primarily in methylotrophic bacteria.11-14 These observations are chemically intriguing because these organisms also encode closely related (and better characterized) CaII-dependent ADHs.15 Understanding how biology selectively recognizes and utilizes LnIIIs in the presence of much more

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2 abundant CaII would have significant biotechnological and industrial impact, given the importance of Lns in numerous current technologies. We have recently demonstrated that Nature has evolved its own LBT to selectively coordinate LnIIIs via adaptation of EF-hand motifs in a protein that we named lanmodulin (LanM, UniprotKB C5B164), from the model methyltroph, Methylobacterium extorquens AM1.16 In the apo form, unusually for EF-hand-containing proteins, LanM possesses little secondary structure, but in the presence of picomolar concentrations of all LnIIIs and YIII it undergoes cooperative folding to a compact conformation with ~50% helical structure. CaII also promotes this conformational change, albeit requiring 108-fold higher metal concentrations than with LnIII. LanM’s four EF-hands are unique in that each contains a proline residue between the first and second putative metal-coordinating residues; a proline residue is almost never encountered at this position in CaMs and other EF-hand-containing proteins.4, 5 Our results suggested that these Pro residues in LanM contribute to decoupling of CaII binding from the conformational change, putatively to suppress CaII responsiveness in the cell. In an effort to understand these unusual properties in molecular detail, here we present the solution structure of LanM complexed with YIII, determined by NMR spectroscopy. This is the first structural analysis of a native Ln-binding protein that is not a PQQ-dependent ADH. Our results reveal a novel architecture for an EF-handcontaining protein and provide insight into how LanM responds to the full range of LnIIIs with high selectivity over CaII. As isolated from M. extorquens, LanM consists of residues 22-133 of the predicted sequence (residues 1-21 are a signal peptide, Figure S1).16 Therefore, we generated a construct for heterologous expression in E. coli comprising an N-terminal Met residue, residues 22-133, and a C-terminal hexahistidine tag (see the Supporting Information for all Experimental Procedures). Mass spectrometric analysis indicates that the first two residues (Met and Ala22) are cleaved during expression (Figure S2). This construct exhibits metal-dependent secondary structural changes and LnIII affinities comparable to untagged, wild-type LanM.16 We chose YIII for structural characterization of metal-bound LanM because it possesses a similar ionic radius and coordination chemistry to the LnIIIs,17, 18 but YIII is diamagnetic, unlike most LnIII ions, facilitating solution structure determination by NMR spectroscopy. Furthermore, YIII-LanM’s apparent dissociation constant (Kd,app), 17 pM, is similar to that observed for physiologically relevant Lns (5 pM for LaIII,


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Biochemistry

3 NdIII, and SmIII).16 Therefore, the structure of YIII-bound LanM is likely representative of the protein bound to any LnIII ion. LanM possesses 4 predicted EF-loops: EF1 (D35-E46), EF2 (D59-E70), EF3 (D84-E95), and EF4 (N108-E119). It binds 3 equivalents of LnIII with picomolar affinity and a fourth with approximately micromolar affinity.16 We previously proposed that the low-affinity site is EF4 because of its Asn rather than Asp as a first residue, a substitution predicted to interfere with metal responsiveness.4 To determine the solution structure of LanM, NMR spectra were collected under conditions in which all four metal binding sites were saturated with YIII. An 1H,15N-heteronuclear single quantum coherence (HSQC) spectrum of the C-terminally tagged protein used for structural analysis is displayed in Figure S3 (the 1H,15N-HSQC spectrum of the N-terminally tagged protein, shown in Figure S4, demonstrates that the location of the tag minimally affects protein structure). Sharp resonance lines were observed for EF1-3, while resonances corresponding to residues P109G113 in EF4 were absent from the spectrum, suggesting chemical exchange on the NMR timescale, as is often observed with micromolar-regime binding. Thus, the features of the YIIIsaturated LanM HSQC spectrum are consistent with weak YIII binding by EF4 compared with the high affinity of the other three EF-hands. The 12 lowest-energy structures of YIII-bound LanM are overlaid in Figure 1A (Figure S5 for backbone trace). Statistics for the NMR refinement are shown in Table 1. Given that LanM was initially identified through its partial co-purification with the Ln-dependent methanol dehydrogenase, XoxF1, Figure S6 shows a surface representation and discusses potential interaction surfaces. Although LanM possesses low identity to characterized EF-hand-containing proteins outside of its 4 EF-loops, we compare the solution structures of YIII-LanM and CaII-CaM (Figure 1B)4, 6 because of their common conformational responsiveness to metal ion binding and shared structural features, despite their distinct overall topologies (vide infra). LanM forms a compact helical bundle, with three central helices forming a hydrophobic core and the four EFloops positioned at the periphery. Unexpectedly, whereas in CaM EF1/EF2 pair to form one globular domain and EF3/EF4 pair to form another,7 LanM’s EF-hands pair as EF1/4 and EF2/3. The helices entering and exiting the metal-binding loops of canonical, 29-residue EF-hands, as in CaM, are contained in the ~25 residues between each EF-loop (Figure S1). However, LanM’s EFloops are only 12-13 residues apart; the structure reveals that this spacing results in fusion of the exiting helix of one EF-hand with the entering helix of the next. The requisite 90° relative


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4 orientation between entering/exiting helices in EF-hands is accomplished in LanM’s compact fold by bifurcation of the helices following EF2 and EF4 by G73 and P123, respectively (Figure S7). In the latter case, P123 allows the final helix to pack to complete the central three-helix bundle. Interestingly, the backbone CO of L131, near the end of this helix, hydrogen bonds with the phenolic OH of Y96 (immediately following EF3), the absorption and fluorescence properties of which are strongly altered by LnIII binding.16 The resulting topology of YIII-bound LanM is, to our knowledge, unique among EF-hand-containing proteins. Table 1. NMR statistics for YIII-bound LanM (PDB ID: 6MI5) for the final ensemble (12 lowest energy structures). Experimental restraintsa Unambiguous NOEs Intraresidue (i = j) Sequential (|i – j| = 1) Medium range (1 < |i – j| < 5) Long range (|i – j| ≥ 5) Total unambiguous NOEs NOEs with multiple assignments Total NOEs Phi/psi angle restraints (TALOS+) Restraint violations NOE distances violated > 0.5 Å Dihedral angles violated > 5° RMSDs from ideal geometry Bond length (Å) Bond angles (°) Impropers Ramachandran plot statistics (%) Most favored regions Additionally allowed regions Generously allowed regions Disallowed regions Coordinate RMSD All residues Backbone atoms 1.8 Heavy atoms 2.3

507 334 252 199 1292 182 1474 192 0 0 0.0014 2.8 0.571 84.4 14.3 1.1 0.2 Ordered 0.7 1.0

aAll

statistics were calculated for residues P23 (the N-terminus of our construct) to R133, excluding the C-terminal His6 tag.


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Figure 1. The solution structure of YIII-LanM reveals significant topology differences with respect to CaII-CaM. (A) Ribbon diagrams representing the 12 lowest-energy models of YIII-LanM. YIII ions are shown in teal spheres, and EF-loops are shown in gray. For clarity, the C-terminal His6tag is not shown. (B) Ribbon diagrams representing the three deposited models of CaII-CaM.7 CaII ions (green spheres) and coordinating residues (sticks) are shown, and the flexible linker is indicated as a dashed line. CaM’s EF3/4 pair is shown in a similar orientation to LanM’s EF2/3


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6 pair in (A); see Figure S7. (C) YIII coordination by the LnIII-binding sites of EF1, EF2, and EF3. Metal-coordinating residues are labeled. Due to intermediate exchange in the 1H,15N-HSQC, YIII was not modeled into EF4, although this site was saturated under the NMR solution conditions. The potentially unique topology of YIII-LanM motivated us to test our model via two additional experiments. In solution, CaII-CaM behaves as two distinct globular domains, connected by a flexible linker.6, 7 Backbone 15N spin-relaxation has been used to characterize the amplitude of this flexibility on the fast (ps-ns) timescale, with the heteronuclear 1H,15N-NOE serving as an especially direct representation of those dynamics. For sites that are internally rigid on this fast timescale, such as those found in secondary structure and ordered loops, the 1H,15N-NOE approaches unity, whereas the measured value approaches zero, or even becomes negative, in highly flexible loops and linkers. The 1H,15N-NOE values for YIII-LanM are uniform and >0.9 for nearly all recorded positions, with the exception of the flexible N-terminus (Figure 2A). In contrast, data for CaII-CaM display a pronounced dip between residues 75-85, indicating that the linker between EF1/2 and EF3/4 is flexible in solution (Figure 2B).6 In summary, our YIII-LanM 1H,15N-NOE

data support the observation of a well-packed hydrophobic core that unites all four

EF-hands into a single globular structure.

Figure 2. Heteronuclear 1H,15N-NOE values for YIII-LanM are consistent with all four EF hands contributing to a single globular domain. (A) YIII-LanM 1H,15N-NOE values are consistently high, supporting the interpretation that it possesses a rigid and compact architecture in solution. (B) 1H,15N-NOE of CaII-CaM, reproduced from ref. 6. reveals a highly flexible linker (residues 7585) separating the N-terminal (EF1/2) and C-terminal (EF3/4) globular domains. Next, we sought an independent method to confirm the pairing of EF1/4 and EF2/3 in YIIILanM. Whereas the proton-proton NOEs used extensively for NMR structure refinement are highly local, the phenomenon of paramagnetic relaxation enhancement (PRE) has the potential to


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7 reveal all amino acid residues found within ~20 Å of an unpaired electron introduced into a protein’s structure.19 In this assay, a loss of 1H,15N-HSQC resonance intensity is observed for all residues near the spin probe. Therefore, we reasoned that introduction of an exogenous electronspin probe into EF1 would induce a strong PRE response in EF4, but not EF2; similarly, labeling of EF3 should induce a strong PRE response in EF2, but not EF4. Cysteine residues were introduced through the point mutations L44C (near EF1) or K93C (near EF3), and these residues were modified with the nitroxide spin label MTSL [1-oxyl-2,2,5,5-tetramethyl-pyrroline-3methyl)methanethiosulfonate]. PRE responses are mapped as a color gradient onto the LanM structure in Figure 3 (raw intensity ratios are displayed in Figure S8). The PRE data confirm quenching of residues near EF4 when L44C is labeled and quenching of residues near EF2 when K93C is labeled. These results are consistent with pairing between EF1/4 and between EF2/3. Together with the continuous and high 1H,15N-NOE baseline that suggests that YIII-LanM forms a single cooperatively folded unit in solution, these experiments strongly support the solution structure presented in Figure 1A.

Figure 3. Paramagnetic relaxation enhancement data support the structural model of YIII-LanM. L44C or K93C LanM were labeled with the cysteine-reactive nitroxide spin label MTSL. (A) Ribbon diagram of YIII-LanM is shown for reference. (B) and (C) Normalized 1H,15N-HSQC resonance intensity changes for each mutant are plotted onto the surface of the lowest energy structure of YIII-LanM, where whiter regions indicate a greater attenuation of signal intensity and darker regions indicate a low-to-absent change in intensity.


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With strong evidence supporting our overall structural model, we turn to the metal binding sites in YIII-LanM. Carboxylate-rich metal binding sites are sparse in 1H-nuclei and therefore yield few NOE constraints. Thus, consistent with standard practice for metal-binding proteins,20, 21 YIII coordination in the EF1-3 binding sites was achieved through model refinement in Amber, in the presence of explicit YIII ions and water molecules (the latter removed prior to PDB deposition) to increase precision in the ligand geometry (see Experimental Methods for details). As a consequence of chemical exchange, only sparse structure constraints were available for EF4 and we did not model a YIII ion into this site. Nevertheless, we note that this fourth site was saturated under our conditions and the determined structure represents a fully YIII-bound form of LanM. In EF1-3, each YIII is coordinated by 4-5 carboxylate sidechains and a backbone carbonyl (Figure 1C). The coordination sphere of YIII in EF3 is shown in detail as a representative example in Figure 4A (see Figure S9 for EF3 for the full 12-member bundle). The consensus sequence for LanM’s EF1-3 is D1-P2-D3-K/N4-D5-G6-T7-I/L8-D9-x10-K/R11-E12, with the underlined residues coordinating the metal ion via side chains or, in the case of T7, a mainchain CO. Although this coordination is broadly similar to CaII binding in canonical EF-loops, the YIII ions in LanM have a higher coordination number (8-9, depending on the site and structure) than does CaII in CaM (7); high coordination numbers are commonly observed in LnIII complexes.18 The higher coordination number in the LanM models appears to result from several factors. First, the conserved D9 directly coordinates in all structures of EF2 and EF3. By contrast, metal coordination by the residue at this position in EF-loops in general is variable (Figure S10); in CaM, for example, the equivalent residue hydrogen bonds to a coordinated solvent molecule rather than directly coordinating the CaII. D9 residues have been shown to increase LnIII-selectivity of a model EF-loop by 100-fold,22 and E9 coordinates the metal ion in the crystal structure of the TbIII-LBT.9 The high conservation in LanM sequences of an Asp at this position or position 11, where it may also be poised for metal coordination, argues for the importance of this additional carboxylate for LnIII binding. Second, in LanM the coordination modes of D3, D5, and even E12 are variable (Figure S9), whereas in CaM, D3 and D/N5 are generally monodentate ligands, and bidentate coordination of E12 is critical for EF-loop function.4 Enhanced EF-loop flexibility was recently also observed in a spectroscopic study of LnIII coordination in CaM.3 Finally, unlike in EF2/3 and despite identical restraints for metal site refinement, YIII in EF1 is not coordinated by D9 in 9 of 12 models. This distinction,


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9 perhaps linked to flexibility in EF4, may explain our biochemical observations suggesting the existence of two sets of high-affinity metal binding sites in LanM.16 Altogether, our structural models suggest that a high density of coordinatively flexible carboxylates, including D9, may be important for LanM to effectively bind the range of biologically relevant LnIII ions (LaIII-SmIII, with ionic radii ranging from 1.30 to 1.22 Å)17 with similar, high affinity.

Figure 4. YIII coordination in EF3 of LanM. (A) YIII is 8- to 9-coordinate, depending on the structure, with ligands provided by conserved carboxylates in the EF-loops and the backbone carbonyl of a conserved Thr. (B) Ni+1–H•••Ni hydrogen bonding in LanM’s EF-hands. The backbone amide hydrogen of D86 forms a hydrogen bond with the backbone nitrogen of P85. A similar interaction is observed in all three YIII-bound EF-loops. An additional aspect of LanM’s EF-hands that is distinct from all other characterized EFhands is the conserved Pro residues at position 2 (P2). We observed putative Ni+1–H•••Ni hydrogen bonds between the amide N of P2 and the amide NH of D3 in EF1-3 (Figure 4B). This type of hydrogen bond has been implicated in catalysis of some prolyl cis-trans isomerizations,23 suggesting a possible rationale for the conservation of this unique amino acid at this position. If formation of this hydrogen bond is important for metal selectivity, it is not clear at present why YIII (and presumably all LnIIIs) would induce it more efficiently than CaII, based on steric considerations.17 It is possible that a lower charge results in CaII binding to a different set of carboxylates than the LnIIIs and YIII, resulting in a distinct EF-loop conformation. Whereas a single


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10 LnIII per EF-hand is sufficient to cause LanM’s conformational change, multiple CaII ions per EFhand seem to be required.16 Clearly, structural characterization of the apo and CaII-bound states of LanM will also be essential for a complete understanding of the basis of the protein’s metal selectivity, and this is a focus of our current efforts. In summary, we have presented the first structural analysis of the highly selective Lnbinding protein, lanmodulin. In its YIII-bound form, this protein adopts a three-helix bundle fold unique among EF hand-containing proteins, which we confirm through heteronuclear NOE and PRE data. YIII-bound LanM’s short α-helices suggest that the protein structure may be poised between coil (apo-LanM) and helical states,24 with metal ion binding shifting the equilibrium to the structure determined here. Understanding the molecular details by which LanM discriminates LnIIIs from CaII and other metal ions to mediate this structural transition will shed light on how methylotrophs use Lns in essential cellular processes and will inspire approaches to selectively detect and accumulate these important metals.

ASSOCIATED CONTENT Supporting Information. Experimental Procedures and Supplementary Tables, Figures, and References. Accession Codes. The chemical shift assignments and coordinates for YIII-LanM have been deposited in the Biological Magnetic Resonance Bank and the Protein Data Bank under accession codes 27604 and 6MI5, respectively. AUTHOR INFORMATION * Corresponding authors. E-mail: [email protected] (J.A.C.), [email protected] (S.A.S.) ORCIDs: Erik C. Cook: 0000-0003-3863-1708 Joseph A. Cotruvo, Jr.: 0000-0003-4243-8257 Scott A. Showalter: 0000-0001-5179-032X Notes. The authors declare no competing financial interests. ACKNOWLEDGEMENTS We thank E.J. Issertell and G. Usher for experimental assistance, T.N. Laremore for assistance with mass spectrometry experiments, and M. Hedglin for instrumentation. J.A.C. gratefully acknowledges the Penn State Department of Chemistry, the Huck Institutes for the Life Sciences, and a Louis Martarano Career Development Professorship for funding.


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11 REFERENCES [1] Wang, C.-L., Aquaron, R. R., Leavis, P. C., and Gergely, J. (1982) Metal-binding properties of calmodulin, Eur. J. Biochem. 124, 7-12. [2] Mulqueen, P., Tingey, J. M., and Horrocks, W. D. J. (1985) Characterization of lanthanide(III) ion binding to calmodulin using luminescence spectroscopy, Biochemistry 24, 6639-6645. [3] Edington, S. C., Gonzalez, A., Middendorf, T. R., Halling, D. B., Aldrich, R. W., and Baiz, C. R. (2018) Coordination to lanthanide ions distorts binding site conformation in calmodulin, Proc. Natl. Acad. Sci. U.S.A. 115, E3126-e3134. [4] Gifford, J. L., Walsh, M. P., and Vogel, H. J. (2007) Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs, Biochem. J. 405, 199221. [5] Halling, D. B., Liebeskind, B. J., Hall, A. W., and Aldrich, R. W. (2016) Conserved properties of individual Ca2+-binding sites in calmodulin, Proc. Natl. Acad. Sci. USA 113, E1216-E1225. [6] Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W., and Bax, A. (1992) Backbone dynamics of calmodulin studied by nitrogen-15 relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible, Biochemistry 31, 5269-5278. [7] Chou, J. J., Li, S., Klee, C. B., and Bax, A. (2001) Solution structure of Ca2+-calmodulin reveals flexible hand-like properties of its domains, Nat. Struct. Biol. 8, 990-996. [8] Burger, D., Cox, J. A., Comte, M., and Stein, E. A. (1984) Sequential conformational changes in calmodulin upon binding of calcium, Biochemistry 23, 1966-1971. [9] Nitz, M., Sherawat, M., Franz, K. J., Peisach, E., Allen, K. N., and Imperiali, B. (2004) Structural origin of the high affinity of a chemically evolved lanthanide-binding peptide, Angew. Chem. Int. Ed. 43, 3682-3685. [10] Allen, K. N., and Imperiali, B. (2010) Lanthanide-tagged proteins - an illuminating partnership, Curr. Opin. Chem. Biol. 14, 247-254. [11] Nakagawa, T., Mitsui, R., Tani, A., Sasa, K., Tashiro, S., Iwama, T., Hayakawa, T., and Kawai, K. (2012) A catalytic role of XoxF1 as La3+-dependent methanol dehydrogenase in Methylobacterium extorquens strain AM1, PLoS ONE 7, e50480. [12] Pol, A., Barends, T. R. M., Dietl, A., Khadem, A. F., Eygensteyn, J., Jetten, M. S. M., and Op den Camp, H. J. M. (2014) Rare earth metals are essential for methanotrophic life in volcanic mudpots, Environ. Microbiol. 16, 255-264. [13] Good, N. M., Vu, H. N., Surlano, C. J., Subuyuj, G. A., Skovran, E., and Martinez-Gomez, N. C. (2016) Pyrroloquinoline quinone ethanol dehydrogenase in Methylobacterium extorquens AM1 extends lanthanide-dependent metabolism to multicarbon substrates, J. Bacteriol. 198, 3109-3118. [14] Wehrmann, M., Billard, P., Martin-Meriadec, A., Zegeye, A., and Klebensberger, J. (2017) Functional role of lanthanides in enzymatic activity and transcriptional regulation of pyrroloquinoline quinone-dependent alcohol dehydrogenases in Pseudomonas putida KT2440, mBio 8, e00570-00517. [15] Anthony, C., and Williams, P. (2003) The structure and mechanism of methanol dehydrogenase, Biochim. Biophys. Acta 1647, 18-23.


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12 [16] Cotruvo, J. A., Jr., Featherston, E. R., Mattocks, J. A., Ho, J. V., and Laremore, T. N. (2018) Lanmodulin: A highly selective lanthanide-binding protein from a lanthanide-utilizing bacterium, J. Am. Chem. Soc., DOI: 10.1021/jacs.1028b09842. [17] Shannon, R. D. (1976) Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides, Acta Cryst. A 32, 751-767. [18] Dehnicke, K., and Greiner, A. (2003) Unusual Complex Chemistry of Rare-Earth Elements: Large Ionic Radii—Small Coordination Numbers, Angew. Chem. Int. Ed. 42, 1340-1354. [19] Battiste, J. L., and Wagner, G. (2000) Utilization of site-directed spin labeling and highresolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data, Biochemistry 39, 5355-5365. [20] Chakravorty, D. K., Wang, B., Lee, C. W., Guerra, A. J., Giedroc, D. P., and Merz, K. M., Jr. (2013) Solution NMR refinement of a metal ion bound protein using metal ion inclusive restrained molecular dynamics methods, J. Biomol. NMR 56, 125-137. [21] Hovey, L., Fowler, C. A., Mahling, R., Lin, Z., Miller, M. S., Marx, D. C., Yoder, J. B., Kim, E. H., Tefft, K. M., Waite, B. C., Feldkamp, M. D., Yu, L., and Shea, M. A. (2017) Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2, Biophys. Chem. 224, 1-19. [22] Drake, S. K., Lee, K. L., and Falke, J. J. (1996) Tuning the equilibrium ion affinity and selectivity of the EF-hand calcium binding motif: substitutions at the gateway position, Biochemistry 35, 6697-6705. [23] Adhikary, R., Zimmermann, J., Liu, J., Forrest, R. P., Janicki, T. D., Dawson, P. E., Corcelli, S. A., and Romesberg, F. E. (2014) Evidence of an unusual N-H•••N hydrogen bond in proteins, J. Am. Chem. Soc. 136, 13474-13477. [24] Sandhu, K. S., and Dash, D. (2007) Dynamic -helices: Conformations that do not conform, Proteins 68, 109-122.


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Biochemistry

13 FOR TABLE OF CONTENTS USE ONLY


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