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Lanmodulin: A highly selective lanthanide-binding protein from a lanthanide-utilizing bacterium Joseph A. Cotruvo, Jr., Emily R. Featherston, Joseph A. Mattocks, Jackson V. Ho, and Tatiana N. Laremore J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b09842 • Publication Date (Web): 14 Oct 2018 Downloaded from http://pubs.acs.org on October 14, 2018

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Journal of the American Chemical Society

Lanmodulin: A highly selective lanthanide-binding protein from a lanthanide-utilizing bacterium Joseph A. Cotruvo, Jr,*,† Emily R. Featherston,† Joseph A. Mattocks,† Jackson V. Ho,† and Tatiana N. Laremore‡ Department of Chemistry, The Pennsylvania State University, University Park, PA 16802 Proteomics and Mass Spectrometry Core, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802 † ‡

Supporting Information Placeholder ABSTRACT: Lanthanides (Lns) have been shown recently to be essential cofactors in certain enzymes in methylotrophic bacteria. Here we identify in the model methylotroph, Methylobacterium extorquens, a highly selective LnIII-binding protein, which we name lanmodulin (LanM). LanM possesses four metal-binding EF-hand motifs, commonly associated with CaII binding proteins. In contrast to other EF hand-containing proteins, however, LanM undergoes a large conformational change from a largely disordered state to a compact, ordered state in response to picomolar concentrations of all LnIII (Ln = La-Lu, Y), whereas it only responds to CaII at near-millimolar concentrations. Mutagenesis of conserved proline residues present in LanM’s EF hands, not encountered in CaII-binding EF hands, to alanine pushes CaII responsiveness into the micromolar concentration range while retaining picomolar LnIII affinity, suggesting that these unique proline residues play a key role in ensuring metal selectivity in vivo. Identification and characterization of LanM provides insights into how biology selectively recognizes low-abundance LnIII over higher-abundance CaII, pointing toward biotechnologies for detecting, sequestering, and separating these technologically important elements.

being best characterized. The specific incorporation of LnIII instead of CaII into the Ln-MDH active site is of great interest from a chemical perspective, given the similar ionic radii of CaII to the biologically relevant early LnIIIs,22 the nearly identical metal coordination environments in the two classes of MDHs,13, 18 and the significantly greater environmental abundance of Ca versus Lns.9 We are working to elucidate the fundamental coordination chemistry principles underlying Nature’s utilization of Lns for essential cellular functions. During our efforts to further characterize the Ln-MDH from M. extorquens, XoxF, we identified a previously uncharacterized LnIII-binding protein that partially co-purified with the Ln-MDH. This protein, which we name lanmodulin (LanM), for lanthanide-modulated protein, contains metal coordination motifs (EF hands) traditionally associated with nanomolar- to millimolar-affinity CaII binding.23 However, LanM responds to LnIIIs with 108fold selectivity over CaII. This manuscript describes the discovery and purification of LanM, characterizes its LnIII binding properties, and probes the basis of its remarkable metal selectivity.

INTRODUCTION Rare earth elements, particularly the lanthanides (Lns), are in high demand as key components in technologies such as permanent magnets, hybrid car batteries, lasers, phosphors, and smartphones.1-2 However, the breadth of the applications of these elements is contrasted with the paucity of economically viable mineable deposits and the difficulty of Ln extraction from ores. The insolubility of Ln salts and similarity of their chemical properties requires harsh, environmentally harmful, and laborious methods for mining and separation.3-5 The technological importance of Lns and the challenges associated with their acquisition have inspired bioengineering methods for more facile and environmentally friendly Ln isolation.6-8 Whereas some plants and microbes have long been known to accumulate these metals,9 only recently has it been established that Lns play an essential biological role, in catalytic activity of certain pyrroloquinoline quinone (PQQ)dependent alcohol dehydrogenase enzymes,10-15 especially methanol dehydrogenases (MDHs). MDHs, found in methylotrophic bacteria, are soluble, periplasmic enzymes that catalyze the oxidation of methanol to formaldehyde, a key metabolic step enabling methanol to serve as the sole carbon source for growth, thereby playing a critical role in the global carbon cycle.16-18 The most extensively characterized MDHs require a CaII ion for activity,18 but recent work has demonstrated the presence of specifically LnIII-dependent MDHs (Ln-MDHs) in several organisms,19-21 with the enzymes from the model methylotroph, Methylobacterium extorquens,12 and Methylacidiphilum fumariolicum SolV13

RESULTS AND DISCUSSION We purified XoxF from M. extorquens AM1 cells grown in the presence of 1 µM LaCl3 and methanol as sole carbon source (see Supporting Information for all experimental details). The protein was purified by ammonium sulfate precipitation, cation exchange chromatography, and size exclusion chromatography (SEC) to high purity (Figure 1a), 1

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and a specific activity of 22 µmol/min/mg at 23 °C (Table S1, Figure S1). After the cation exchange step, we were struck by the

Figure 1. LanM is a novel EF hand-containing protein. A) SDS-PAGE analysis of XoxF purification and copurifying band. Lane 1: Molecular weight marker. Lane 2: M. extorquens crude extract. Lane 3: XoxF after cation exchange chromatography (highlighted band was excised and analyzed by mass spectrometry, revealing LanM). Lane 4: Purified XoxF. Lane 5: Heterologously expressed and purified LanM. B) Sequences of the EF hands of LanM and of human CaM (as a representative canonical EF hand-containing protein). In CaM, residues providing sidechain CaII ligation are shown in red, and residues involved in a hydrogen bonding network to a metal-coordinated water molecule are shown in green; in LanM, aligning residues are shown in blue, and the unique Pro residues are bolded in purple.

example, CaM comprises two pairs of EF hands, which cooperatively bind 4 CaII ions,28 inducing a conformational change to promote binding to target proteins.29-30 In most EF hands,23, 26 the CaII ions are 7-coordinate: the 1st, 3rd, 5th, and 12th positions of the motif provide side chain oxygen ligands, the 7th position provides a main-chain carbonyl oxygen, and the 9th position either directly coordinates the CaII or, more typically, hydrogen bonds to a coordinated solvent molecule (Figure 1b). EF hands also bind LnIIIs, often more tightly than CaII (e.g., ~1 nM for TbIII-CaM31 vs. ~1 µM for CaII-CaM32), although LnIII binding is not physiologically relevant for these proteins. LanM’s EF hands possess several unique features relative to canonical EF hands. First, LanM retains all of the metalbinding Asp and Asn residues present in typical EF hands but also features an Asp residue in the 9th position in each of its EF hands, whereas Asp is encountered at this position in roughly one-third of EF hand sequences.23 An Asp residue at position 9 has been shown in a model EF hand to contribute ~2 orders of magnitude selectivity for LnIII over CaII.33-34 Second, Asn is rarely if ever observed at the first position in functional EF hands,23, 26 as it is in EF-hand 4 (EF4) in LanM. Third, all of LanM’s EF hands also possess a Pro residue at the 2nd position, a highly uncommon feature of EF hands in general, and encountered in only