Modularity in Protein Evolution: Modular Organization and De Novo Domain Evolution in Mollusk Metallothioneins Sara Calatayud,1 Mario Garcia-Risco,2 Veronika Pedrini-Martha,3 Douglas J. Eernisse,4 Reinhard Dallinger,3  Oscar Palacios,2 Mercè Capdevila,2 and Ricard Albalat* ,1 1 Departament de Genètica, Microbiologia i Estadıstica and Institut de Recerca de la Biodiversitat (IRBio), Facultat de Biologia, Universitat de Barcelona, Barcelona, Catalonia, Spain 2 Departament de Quımica, Facultat de Ciències, Universitat Autonoma de Barcelona, Cerdanyola del Vallès, Spain 3 Department of Zoology and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria 4 Department of Biological Science, California State University Fullerton, Fullerton, CA *Corresponding author: E-mail: ralbalat@ub.edu. Associate editor: Yoko Satta Abstract Metallothioneins (MTs) are proteins devoted to the control of metal homeostasis and detoxification, and therefore, MTs have been crucial for the adaptation of the living beings to variable situations of metal bioavailability. The evolution of MTs is, however, not yet fully understood, and to provide new insights into it, we have investigated the MTs in the diverse classes of Mollusks. We have shown that most molluskan MTs are bimodular proteins that combine six domains—a, b1, b2, b3, c, and d—in a lineage-specific manner. We have functionally characterized the Neritimorpha b3b1 and the Patellogastropoda cb1 MTs, demonstrating the metal-binding capacity of the new c domain. Our results have revealed a modular organization of mollusk MT, whose evolution has been impacted by duplication, loss, and de novo emergence of domains. MTs represent a paradigmatic example of modular evolution probably driven by the structural and functional requirements of metal binding. Key words: a, b1, b2, b3, c, and d domains, bi- and multimodular metallothioneins, cysteine motifs, de novo evolution, metal-binding capacity and preference. Introduction 2016; Nam and Kim 2017), and not on their N- or C-terminal position. Metallothioneins (MTs) are a superfamily of intracellular, Thanks to their metal-binding capacity, MTs are directly cysteine-rich (15–30%), and mostly low-molecular-weight involved in metal homeostasis and detoxification, but also in Article (<100 amino acids) proteins present across eukaryotes, from radical scavenging, oxidative stress protection, and antiapop- various protists to plants, fungi, and animals (Capdevila and totic defense (Capdevila et al. 2012), leading to their role as a Atrian 2011; Blindauer 2014). Their cysteine (Cys, C) residues model system for the investigation of the genetic mechanisms are arranged in distinctive motifs (i.e., CxC, CC, and CCC), by which organisms adapt to diverse metal bioavailabilities or whose number and distribution led to the original definition stress. For instance, duplications of MT genes (Maroni et al. of the two functional domains in vertebrate MTs (Braun et al. 1987; Adamo et al. 2012) or expansions of MT domains 1986) designated as a domain (with 11–12 cysteines at the C- (Tanguy and Moraga 2001; Jenny et al. 2016; Pedrini- terminal region) and b domain (with nine cysteines at the N- Martha et al. 2020), along with elevated levels of MT expres- terminal region), joined by a linker sequence. In this bimod- sion (Timmermans et al. 2005; Janssens et al. 2008, 2009; Costa ular structure made of two domains, the cysteine motifs of et al. 2012; Catalan et al. 2016; de Francisco et al. 2018) or each domain are able to form metallic clusters, thus confer- changed metal specificity (Tio et al. 2004; Palacios et al. 2011; ring the capacity of binding both essential and nonessential de Francisco et al. 2017) have been considered adaptive metals (Nielson and Winge 1985). Notice that the a and b events contributing to increase the metal and stress tolerance nomenclature has been also used for referring to N-terminal of the organisms in different environments. In particular, a (a) or C-terminal (b) domains in some gastropod MTs previous thorough analysis investigating the role of cadmium (Baumann et al. 2017; Niederwanger, Calatayud, et al. 2017; (Cd) on the evolution of gastropod MTs has led us to suggest Palacios et al. 2017; Schmielau et al. 2019). In order to avoid that lineage-specific changes of metal-selectivity features confusion, however, we will use here the a/b nomenclature in might have been important during the recurrent colonization its primary meaning for classifying domains based on the of marine gastropods to terrestrial and freshwater habitats, number and distribution of cysteine motifs (Jenny et al. where they had to face the challenge of adapting to ß The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Open Access 424 Mol. Biol. Evol. 38(2):424–436 doi:10.1093/molbev/msaa230 Advance Access publication September 11, 2020 Modularity in Protein Evolution . doi:10.1093/molbev/msaa230 MBE Table 1. Cysteine Motifs of Mollusca Bimodular MTs. Class Clade Domain Cysteine Motifs N-Terminal Linker C-Terminal Conchifera Gastropoda Patellogastropoda c b1 CCx5CCx4CCx6CCx7CC X5–6 [CxC]x3[CxC]x3Cx5[CxC]x3[CxC] Vetigastropoda b3 b1 Cx3Cx4[CxC]x3[CxC]x4[CxC]x2C X3–6 [CxC]x3[CxC]x3Cx5[CxC]x3[CxC] Neritimorpha b3 b1 Cx3Cx5[CxC]x3[CxC]x3[CxC]x2C X3–4 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] Caenogastropoda b3 b1 Cx3Cx4[CxC]x5[CxC]x3[CxC]x2C X2–3 [CxC]x4[CxC]x3Cx6[CxC]x3[CxC] Heterobranchia b3 b1 Cx3Cx4[CxC]x3[CxC]x3[CxC]x2C X2 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] Scaphododa b2 b1 [CxC]x5[CxC]x3[CxC]x4[CxC]x4C X8–10 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] Bivalvia a b1 [CxC]x5[CxC]x3Cx5[CxC]x3[CxC]x3[CxC]x2C X3–4 [CxC]x6[CxC]x3Cx5[CxC]x3[CxC] b2 b2 [CxC]x5[CxC]x3[CxC]x4[CxC]x2C X2–3 Cx2Cx4[CxC]x3[CxC]x3[CxC]x2C b2 like b1 Cx4Cx4[Cx0–1C]x4[Cx0–1C]x3[CxC]x2C X1–3 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] Cephalopoda a b1 [CxC]x6[CxC]x3Cx4[CxC]x3[CxC]x3[CxC]x2C X3–4 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] Monoplacophora b2 b1 [CxC]x6[CxC]x3[CxC]x2Cx[CxC]x2C X3 [CxC]x3[CxC]x3Cx5[CxC]x3[CxC] Aculifera Polyplacophora a b1 [CxC]x4[CxC]x3Cx4[CxC]x3[CxC]x3[CxC]x2C X3 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] Solenogastres b2 b1 [CxC]x5[CxC]x3[CxC]x4[CxC]x4C X6 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] a b1 [CxC]x5[CxC]x3Cx5[CxC]x3[CxC]x3[CxC]x2C X7 [CxC]x3[CxC]x3Cx5[CxC]x3[CxC] Caudofoveata d b1 [CxC]x3[CxC]CCx4Cx3[CxC]x3CCx4[CxC]C X3 [CxC]x4[CxC]x3Cx5[CxC]x3[CxC] ecosystems with different levels of metal bioavailability Heterobranchia) and from all mollusk classes, identifying (Dallinger et al. 2020). In this context, studies on MT evolu- and classifying new MTs from other conchiferan (i.e., tion have been of interest to evolutionary ecologists (Janssens Gastropoda, Bivalvia, Cephalopoda, Monoplacophora, and et al. 2009; Faddeeva-Vakhrusheva et al. 2016; Zhang et al. Scaphopoda) and aculiferan (i.e., Polyplacophora, 2018, 2019; Purac et al. 2019) who have associated environ- Solenogastres, and Caudofoveata classes) mollusks. In sum- mental factors—that is, concentrations of heavy metals— mary, by collecting 272 MTs from 189 different species, we with the evolution of diverse MTs. have created the most comprehensive catalog of mollusk Gastropoda is composed of five distinctive lineages ranked MTs compiled so far, and we have exposed patterns of mod- as subclasses, whose interrelationships are still controversial: ular organization across molluskan MTs. Patellogastropoda, Vetigastropoda, Neritimorpha, Caenogastropoda, and Heterobranchia (Zapata et al. 2014; Cunha and Giribet 2019). Although several gastropod MTs Results have been previously investigated, these studies have an un- Identification of Patellogastropoda MTs even phylogenetic distribution. MT identification and metal- We surveyed the genome project of the L. gigantea binding selectivity have been determined for 15 MTs from (Patellogastropoda: Lottiidae). This search yielded an auto- seven heterobranch species (with the most extensive studies), matically predicted MT (Gene ID: 20249168; hypothetical for four MTs from three caenogastropod species, and for a protein: XP_009056965.1), whose sequence, structure, and single vetigastropod MT. In order to fully understand the size suspiciously differed from other Gastropoda MT genes. evolution of gastropod MTs and their metal-binding specific- The low-quality sequence of the genomic region containing ities, it was therefore necessary to extend studies to include the putative MT gene (NW_008709190.1) prompted us to particular species of Patellogastropoda (true limpets) and reanalyze this region by polymerase chain reaction (PCR) am- Neritimorpha (snails known as nerites and their allies). To plifying, cloning, and resequencing >12 kb of L. gigantea ge- add both patellogastropods and neritimorphs here, we have nome (supplementary fig. S1A, Supplementary Material conducted an exhaustive survey of gastropod MTs in public online). Sequence analysis identified two MT genes tandem databases, including raw sequencing data from transcrip- repeated in this region (accession number MK795721), which tomic and genomic high-throughput sequencing projects. we named LgiMT1 and LgiMT2. Our predictions were further We have been able to reconstruct and identify new MTs supported by PCR amplification of the corresponding cDNAs from limpet and neritid species. We have characterized the (MK770430 and MK770431) and by nine L. gigantea metal-binding properties of new MTs for each, selecting Lottia expressed sequence tags (supplementary table S1, gigantea and Nerita peloronta MTs as representatives of each Supplementary Material online), corroborating the misassem- taxon. Our data set extends the knowledge of metal-binding bly of the MT genomic region in the genome project, and preferences to MTs of previously unstudied gastropod taxa, revealing some degree of polymorphism at the amino acid broadening the scope of known diversification of metal- level (S/T and S/A at amino acid positions 3 and 68, respec- binding selectivity in MTs for gastropods, the most species- tively, supplementary fig. S1B, Supplementary Material on- rich, and arguably the most morphologically and ecologically line). Comparison of the coding region (CDS) of LgiMT1 diverse, class of Mollusca. and LgiMT2 genes revealed that they were 97.78% identical By extending what we have learned with new genomic at the nucleotide level (220 out of 225 nt) and both encoded searches, we have added novel MTs from the other three for a 75 amino acid protein with 19 cysteines (25.7%). LgiMT1 gastropod clades (Vetigastropoda, Caenogastropoda, and and LgiMT2 proteins only differed at positions 6, 20, and 49: P, 425 Calatayud et al. . doi:10.1093/molbev/msaa230 MBE FIG. 1. Structural evolution of MTs along the Mollusca phylum. Most mollusk MTs have a bimodular structure made of a variable taxon-specific N- terminal domain—a, b2, b3, c or d—, and a conserved C-terminal b1 domain. Domains are classified based on the number and configuration of the cysteine motifs. Based on the distribution of domains among the different clades, the most parsimonious evolutionary scenario would be that a, b1, and b2 domains are ancient, already present in the MTs of mollusk ancestor. In contrast, b3, c, and d domains would be de novo domains arose in Gastropoda, Patellogastropoda, and Caudofoveata, respectively. Domain gains (circled) and losses (crossed) are indicated under each clade. Deviations of the standard bimodular structure are found in MTs of Bivalvia and Gastropoda, including multimodular MTs with more than two domains (an [Jenny et al. 2004, 2016]; b1n and b2-liken [Nam and Kim 2017] in Bivalvia; and b3n in Heterobranchia and Caenogastropoda [Niederwanger, Calatayud, et al. 2017; Palacios et al. 2017]), and MTs lacking the conserved b1 domain (an and b2b2 MTs in Bivalvia; b3n MTs in Heterobranchia). Phylogenetic relationships within Mollusca are based on Zapata et al. (2014), Cunha and Giribet (2019), and Kocot et al. (2020) L, and P residues in LgiMT1, and A, S, and S in LgiMT2 (sup- that this c domain is not related with the N-terminal, six Cys plementary fig. S1B, Supplementary Material online). domain of plant Ec-1 MTs, also named c domain [Loebus et al. We extended the limpet MT listing to three families and 2011]), which was connected by a linker of four to six amino eight additional species of Patellogastropoda (supplementary acids to an archetypal 9-Cys b1 domain fig. S2A and table S2, Supplementary Material online, and ([CxC]x3[CxC]x3Cx5[CxC]x3[CxC]) (Jenny et al. 2016; Nam Dallinger et al. 2020). These include five species of Lottiidae: and Kim 2017) at the C-terminal region (table 1 and supple- Lottia digitalis (LdiMT1 partial), Lottia kogamogai (LkoMT1), mentary fig. S2A, Supplementary Material online). We con- Lottia scutum (LscMT1 partial), Nipponacmea fuscoviridis cluded therefore that the structure of Patellogastropoda MTs (NfuMT1), and Patelloida pygmaea (PpyMT1); two species was of c/b1 domains (fig. 1). of Nacellidae: Cellana rota (CroMT1) and Nacella concinna (NcoMT1); and one species of Patellidae: Patella vulgata Metal-Binging Capacity of Patellogastropoda MTs (PvuMT1 and PvuMT2). Amino acid comparison of the 11 In order to demonstrate the MT nature and to explore the Patellogastropoda MTs revealed high sequence similarity metal-selectivity features of the two L. gigantea MTs, we stud- (from 69% to 97% identity) with the distinctive 19 cysteines ied the formation of metal-LgiMT1 and metal-LgiMT2 com- fully conserved. Interestingly, the 19 cysteines were organized plexes by the proteins heterologously expressed in Escherichia into two domains: a novel MT domain at the N-terminal coli and grown in media supplemented with copper (Cu), region with ten cysteines arranged in five CC pairs cadmium (Cd), or zinc (Zn) salts by inductively coupled (CCx5CCx4CCx6CCx7CC) that we named c domain (notice plasma atomic emission spectrometer (ICP-AES) and 426 Modularity in Protein Evolution . doi:10.1093/molbev/msaa230 MBE FIG. 2. Deconvoluted ESI-MS spectra LgiMT1 (A), LgiMT2 (B), and cLgiMT2 (C) recombinantly produced by Escherichia coli in Cd-enriched media. electrospray ionization mass spectrometry (ESI-MS) analyses. metal-cLgiMT2 preparations obtained after purification ICP-AES is an analytical technique that allows for protein were characterized by ICP-AES and ESI-MS. The results quantification and metal-to-protein stoichiometry determi- showed a clear preference of cLgiMT2 for divalent Zn(II) nation through the measurement of element composition of and Cd(II) ions, as major M4-cLgiMT2 species were identified the samples (S, Zn, Cd, and Cu) (Bongers et al. 1988), and ESI- for both metal ions by ESI-MS at pH 7. Interestingly, forma- MS is used to determine the molecular mass of the species tion of Cd4-cLgiMT2 complexes was more favored than Zn4- formed, that is, the speciation of the samples (Capdevila et al. cLgiMT2 complexes, as suggested by the fact that the former 2012). The ICP-AES and native ESI-MS (recorded at neutral one was obtained as a single species (fig. 2C), whereas the pH in order to allow the observation of unaltered species) latter one coexisted with other minor species (data not analyses (data not shown) of the recovered samples showed shown). This Cd4-cLgiMT2 cluster also exhibited a high resis- that LgiMT1 and LgiMT2 rendered a mixture of metallated tance against demetallation at acidic pH levels, as this re- species both in Zn(II)- as well as in Cu(II)-supplemented cul- quired an acidification down to pH 1 to obtain the apo- tures, thus indicating the absence of preference for either of protein. Conversely to the Zn- and Cd-cultures, the prepara- those metal ions (Zn(II) and Cu(II) stand for divalent Zn2þ tions obtained from Cu(II)-enriched media yielded mixtures and Cu2þ ions). On the contrary, unique Cd7-LgiMT1 and of multiple Cux-cLgiMT2 complexes (x ranging from 5 to 10), Cd7-LgiMT2 species, that is, MTs loaded with seven Cd(II) confirming the poor preference of this domain for Cu(I). ions, were recovered from Cd-enriched culture media (fig. 2A and B). The identification of single species after re- combinant metal-supplemented productions normally Identification of Neritimorpha MTs reveals the existence of a preferred thermodynamically fa- We collected 12 MTs from two families and six species of vored metal cluster, whereas in the absence of any specific Neritimorpha, including five species of Neritidae: Clithon ret- species, the formation of a variety of species with similar but ropictum (CretrMT1 and CretrMT2), Nerita albicilla (NalMT1 not identical metal content is observed (Palacios et al. 2014). and NalMT2), Nerita melanotragus (NmeMT1 and The observation of single Cd7-LgiMT species, together with NmeMT2), N. peloronta (NpeMT1 and NpeMT2), and the observation by acid ESI-MS (recorded at pH 2.4 that lead Neritina pulligera (NpuMT1 and NpuMT2); one species of to partial protonation of thiol Cys groups, which normally Neritopsidae: Titiscania limacina (TliMT1 and TliMT2 partial) results on the release of Zn and Cd while bound Cu is main- (supplementary fig. S2B and table S2, Supplementary Material tained) that both proteins are reluctant to release Cd(II) by online, and Dallinger et al. 2020). All Neritimorpha species acidification at pH 2.4 giving rise to Cd4-LgiMT species, dem- possessed two MTs, and this appears to be a lineage-specific onstrated the Cd-thionein character of both gene duplication shared by at least two of the four extant Patellogastropoda MTs. superfamilies within Neritimorpha. As for patellogastropods, amino acid comparison of the 12 Neritimorpha MTs showed Metal-Binding Functionality of the New c Domain high sequence conservation (from 65% to 96% identity) with In order to determine the independent metal-binding capac- 19–20 cysteines organized in two MT domains: at the N-ter- ity of the new c domain, we analyzed its ability to form metal minal region, a 9-Cys b3 domain (Cx3Cx4– complexes when expressed alone. Thus, we heterologously 5[CxC]x3[CxC]x3[CxC]x2C, formerly known as a1/2 domain expressed the 10-Cys c domain of LgiMT2 (from Met1 to in non-Neritimorpha species [Baumann et al. 2017; Gln45), hereafter as cLgiMT2, in E. coli grown in medium Niederwanger, Calatayud et al. 2017; Palacios et al. 2017; supplemented with Cu, Cd, or Zn salts. The election of the Schmielau et al. 2019] with an extra cysteine in c domain of LgiMT2 relied on the fact that it was more con- Neritimorpha MT1s), connected by a linker of three to four served than that of LgiMT1 when compared across the c amino acids to a 9-Cys b1 domain at the C-terminal region domains of other Patellogastropoda MTs (supplementary ([CxC]x3–4[CxC]x3Cx5[CxC]x3[CxC]) (table 1 and supple- fig. S2A, Supplementary Material online). The distinct mentary fig. S2B, Supplementary Material online). The 427 Calatayud et al. . doi:10.1093/molbev/msaa230 MBE FIG. 3. Deconvoluted ESI-MS spectra of the in vivo preparations of Escherichia coli recombinant NpeMT1 obtained from a Zn-enriched medium (A) and NpeMT2 synthesized in Cd-enriched cultures (B). structure of Neritimorpha MTs was therefore of b3/b1 terminal 9-Cys b3 domain (Cx3Cx4[CxC]x3– domains (fig. 1). 5[CxC]x3[CxC]x2C) linked by two to three residues to a C- terminal 9-Cys b1 domain ([CxC]x3–4[CxC]x3Cx5– Metal-Binding Capacity of Neritimorpha MTs 6[CxC]x3[CxC]) (table 1 and supplementary fig. S2C, In order to analyze the metal-binding abilities of Supplementary Material online). The archetypal structure of Neritimorpha MTs, we studied the formation of metal-MT Caenogastropoda MTs was therefore of b3/b1 domains complexes of NpeMT1 and NpeMT2 heterologously (fig. 1). Remarkably, b3 domain duplications were observed expressed in E. coli grown in medium supplemented with in a number of Caenogastropoda MTs: one single duplication Cu, Cd, or Zn salts. Metal-NpeMT complexes were purified (i.e., b3.1/b3.2) in 13 MTs; two duplications (i.e., b3.1/b3.2/ and analyzed by ICP-AES and ESI-MS. Our data showed that b3.3) in JjaMT1, PbrMT2, and PcanMT2 sequences; three both Neritimorpha MTs presented similar specificities for di- duplications (from b3.1 to b3.4) in partial EheMT2 and valent metal ions, Zn(II) and Cd(II). However, their binding PcanMT1; five duplications (from b3.1 to b3.6) in preference was not exactly the same, since NpeMT1 was AplaMT2; and six duplications (from b3.1 to b3.7) in more specific for Zn(II) than NpeMT2, whereas NpeMT2 McornMT2 (fig. 4A and supplementary fig. S3A, was more specific for Cd(II) than NpeMT1. Consequently, Supplementary Material online). Since these multi-b3 MTs Zn6-NpeMT1 and Cd6-NpeMT2 complexes could be recov- were unevenly distributed among the Littorinimorpha, ered as single species (fig. 3). The Zn-thionein character of Neogastropoda, and “architaenioglossan” representatives, in- NpeMT1 and the Cd-thionein nature of NepMT2 were also dependent events of internal domain duplications were the supported by the recombinant productions of this protein in most plausible origin of such MTs (Schmielau et al. 2019). Cu-enriched media, which rendered mixtures of heterome- Also noteworthy, ten MTs (most of them duplicated copies) tallic Zn, Cu-MT complexes for NpeMT1, but mixtures of of the Architaenioglossa order showed an additional H3–4C4 homometallic Cu-MT species for NpeMT2 (data not shown). motif (HxHHHx2Cx3Cx6–9Cx0–1C) at the N-terminal region (supplementary fig. S2C, Supplementary Material online). MTs from Other Gastropoda Clades Since these MTs belonged to the same taxonomic group, a We extended the surveys of MTs to gastropod species be- lineage-specific event that added the H3–4C4 motif to the N- longing to Caenogastropoda, Heterobranchia, and terminus of an ancestral Architaenioglossa MT duplicate Vetigastropoda, considered separately below. We collected would be the most parsimonious explanation. a total of 163 MT sequences, including newly identified MTs and previously reported ones (supplementary table S2, Heterobranchia Supplementary Material online). From an MT perspective, this was the most studied Gastropoda clade. We collected 86 MT sequences (61 new) Caenogastropoda MTs of 55 Heterobranchia species. MT multiplicity was observed in We collected 55 MT sequences (43 new) of 43 22 species with two (14 species), three (seven species), or four Caenogastropoda species (supplementary fig. S2C and table (one species) MTs (supplementary fig. S2D and table S2, S2, Supplementary Material online). MT multiplicity was ob- Supplementary Material online). Size diversity of served in 11 species (ten species with two, and Pomacea Heterobranchia MTs was the highest within the canaliculata with three MTs). Size diversity within Gastropoda class, with MTs ranging from 58 (AbaMT1, Caenogastropoda was high, with MTs ranging from 66 amino GscMT1, GcuMT1, GtrMT1, and LstMT1) to 319 (AbiMT4) acids (BacMT1) to 251 amino acids (McornMT2), with a cys- amino acids, with a cysteine content of 15 (25%) and 85 teine content of 17 (25.8%) and 72 cysteines (28.7%), respec- cysteines (26.2%), respectively. Overall, Heterobranchia MTs tively. Overall, Caenogastropoda MTs were organized in an N- were organized in a N-terminal 9-Cys b3 domain 428 Modularity in Protein Evolution . doi:10.1093/molbev/msaa230 MBE FIG. 4. Schematic representation of multimodular Gastropoda MTs. Internal duplications of the N-terminal b3 domain (gray box) generated multimodular MTs in Caenogastropoda (A) and Heterobranchia (B) (see supplementary fig. S2 and table S2, Supplementary Material online, for further details of species). A variable number of b3 domains, ranging from 1 (top) to 9 (bottom), is followed the by a conserved b1 domain (black box). In Heterobranchia, Biomphalaria MTs have three b3 domains followed by a C-terminal tail of five cysteines (C-term) but lack the conserved b1 domain. (Cx3Cx4[CxC]x3[CxC]x3[CxC]x2C) linked by two residues to a and supplementary fig. S2E, Supplementary Material online). C-terminal 9-Cys b1 domain ([CxC]x4[CxC]x3Cx5[CxC]x3 The last common ancestor for Vetigastropoda thus likely had [CxC]) (table 1 and supplementary fig. S2D, Supplementary MTs with b3/b1 domains (fig. 1). Material online). The structure of Heterobranchia MTs was Comparisons across all of the phylogenetically diverse therefore of b3/b1 domains (fig. 1). There were, however, Gastropoda have suggested that the ancestral Gastropoda several exceptions to this structure. Species of Lymnaeidae, already had a b3/b1 MT (fig. 1), which underwent diverse for instance, lacked the last [CxC] motif of the b1 domain, lineage-specific modifications during evolution: 1) gene dupli- MTs of Bradybaena similaris and Fiona pinnata had an addi- cations leading to parallel MT multiplicity in divergent gas- tional b3 domain (i.e., b3.1/b3.2) (supplementary fig. S2D, tropod lineages, 2) internal domain duplications and losses in Supplementary Material online), and Alinda biplicata the Caenogastropoda and Heterobranchia taxa, and 3) AbiMT3 and AbiMT4 had eight and nine b3 domains, respec- acquisitions of novel modules such the H3–4-C4 motif in cer- tively (Pedrini-Martha et al. 2020). But the most deviant tain Caenogastropoda, and the new c domain in Heterobranchia MTs were those of the Biomphalaria species, Patellogastropoda. B. glabrata and B. pfeifferi. These MTs lacked the b1 domain and had three b3 domains (b3.1/b3.2/b3.3) followed by a C- New MTs in Other Mollusk Classes terminal tail with five cysteines (Cx6Cx5Cx4CC) (fig. 4B and Recent phylogenomic analyses have split Mollusks into two supplementary fig. S3B, Supplementary Material online) major clades (see fig. 1) (Kocot et al. 2011, 2020; Smith et al. (Niederwanger, Calatayud, et al. 2017). The fact that both 2011): Conchifera (including Gastropoda, Bivalvia, species shared the same structure suggested that it could Cephalopoda, Monoplacophora, and Scaphopoda) and be a general MT feature for Biomphalaria. Aculifera (including Polyplacophora, Solenogastres, and Caudofoveata). We used publicly available molluskan Vetigastropoda Sequence Read Archives (SRA) projects to assemble new We collected 22 MT sequences (18 new) of 18 Vetigastropoda MTs that, together with previously reported ones, repre- species (supplementary fig. S2E and table S2, Supplementary sented an extensive evolutionary list of mollusk MTs. Thus, Material online). MT multiplicity was found in four species: our analysis has led us to identify for the first time MTs in Clanculus pharaonius (CphMT1 and CphMT2 partial), Scaphopoda, Cephalopoda, and Monoplacophora classes, Perotrochus lucaya (PluMT1 and PluMT2), Phasianella ventri- and the first MTs for chiton, solenogastres and caudofoveatan cosa (PveMT1 and PveMT2), and Prothalotia lehmanni representatives of Aculifera. (PleMT1 and PleMT2). Overall, Vetigastropoda MTs were 65–71 amino acid long with 18 cysteines (25.4–27.7%) orga- Conchifera Clade nized in a N-terminal 9-Cys b3 domain (Cx3Cx4[CxC]x3 Scaphopoda. This molluskan class has been considered the [CxC]x3–4[CxC]x2C) linked by 3–6 residues to a C-terminal sister group of Gastropods (Smith et al. 2011), though this 9-Cys b1 domain ([CxC]x3[CxC]x3Cx5[CxC]x3[CxC]) (table 1 phylogenetic relationship is still under debate. We identified 429 Calatayud et al. . doi:10.1093/molbev/msaa230 MBE FIG. 5. Hypothetical relationship between a and b domains. Based on the configuration of the C and [CxC] motifs, a putative connection between a and b domains is hypothesized. The five [CxC] motifs and the two additional Cs distributed as [CxC]2-C-[CxC]3-C in the a domain appear to have been trimmed (shadowed) at the C-terminal end in b1 domain: [CxC]2-C-[CxC]2; at the middle in b2: [CxC]4-C; or at the N-terminal end in b3: C- C-[CxC]3-C. two Scaphopoda MTs from Antalis entalis (AenMT1) and and the oyster (Alectryonella plicatula) ApliMT1: a/b1/b1; in Graptacme eborea (GebMT1) species (supplementary fig. Crassostrea MTIIs: an or a/b1/b1; or in the scallop S2F and table S2, Supplementary Material online). (Argopecten irradians) AirMT1 and AirMT2: b2-like/b2-like/ Scaphopoda MTs were 68–72 amino acid long with 18–19 b2-like/b1 and b2-like/b2-like/b1, respectively (notice that cysteines (26.4%) organized in a N-terminal 9-Cys b domain organization of the [CxC] motifs in the b2-like domain was ([CxC]x5[CxC]x3[CxC]x2–4[CxC]x4C) linked by 8–10 residues more similar to b3 than to b2 domain) (Tanguy and Moraga to a C-terminal 9-Cys b1 domain ([CxC]x4[CxC]x3 2001; Baek et al. 2009; Jenny et al. 2016; Nam and Kim 2017). Cx5[CxC]x3[CxC]) (table 1 and supplementary fig. S2F, Cephalopoda. We identified the MTs of four Cephalopoda Supplementary Material online). Interestingly, the organiza- species: Nautilus pompilius (NpoMT1), Octopus vulgaris tion of the [CxC] motifs in the Scaphopoda N-terminal b (OvuMT1), Octopus bimaculoides (ObiMT1), and Sepia escu- domain, [CxC]4-C, resembled that of b2 domain defined in lenta (SesMT1) (supplementary fig. S2H and table S2, the unconventional bivalve MTIIIs (Jenny et al. 2016), whereas Supplementary Material online). Cephalopoda MTs were it differed from that of Gastropoda b1 and b3 domains— 69–73 amino acid long with 19–21 cysteines (27.5–28.7%) [CxC]2-C-[CxC]2 and C-C-[CxC]3-C, respectively—(table 1 organized in a/b1 domains more similar to those of other and fig. 5). The structure of Scaphopoda MTs was therefore molluskan classes in the Nautilus MT than in the other spe- of b2/b1 domains (fig. 1). cies. The N-terminal 12-Cys a domain ([CxC]x5– Bivalvia. In order to have a broad perspective of the MTs 6[CxC]x3Cx4[CxC]x3[CxC]x3[CxC]x2C) in the Nautilus MT, within Bivalvia, we analyzed 62 MTs from 36 species (supple- but slightly divergent in the coleoid cephalopods ([CxC]x5– mentary fig. S2G and table S2, Supplementary Material on- 6[CxC]x3Cx4Cx3[CxC]x3[CxC]x2C in Sepia and Octopus MTs), line). For clarity, and since the structural diversity of MTs in was linked by three or four residues to a C-terminal 9-Cys b1 oysters and mussels has been extensively described elsewhere domain ([CxC]x4[CxC]x3Cx5[CxC]x3[CxC] in Nautilus and (Mackay et al. 1993; Jenny et al. 2004, 2016; Aceto et al. 2011), Sepia MTs, but somewhat modified in Octopus MTs; table 1 we focused our analysis on the MTI and MTIV within avail- and supplementary fig. S2H, Supplementary Material online). able species of Ostreidae, and on MT10, MT10B, and MT20 Based on Nautilus and Sepia MTs, we concluded that the within Mytilidae, which have been considered to have the prototypical structure of Cephalopoda MTs was of a/b1 plesiomorphic condition for bivalve MTs (Nam and Kim domains (fig. 1). 2017). Most Bivalvia MTs were 66–78 amino acid long with Monoplacophora. We identified the first MT in a 21–25 cysteines (26.9–32%) organized in an N-terminal 12- Monoplacophora species, Laevipilina hyalina (LhyMT1) (sup- Cys a domain ([CxC]x5[CxC]x3Cx4–5[CxC]x3[CxC]x3– plementary fig. S2I and table S2, Supplementary Material on- 4[CxC]x2C) linked by three residues to a C-terminal 9-Cys line). LhyMT1 was 63 amino acid long with 19 cysteines b1 domain ([CxC]x3–6[CxC]x3Cx5[CxC]x3–5[CxC]) (table 1 (30.2%) organized in an N-terminal b2 domain and supplementary fig. S2G, Supplementary Material online). ([CxC]x6[CxC]x3[CxC]x2[CxC]xCx2C) linked by three residues The likely ancestral structure of these Bivalvia MTs was there- to a C-terminal 9-Cys b1 domain ([CxC]x3[CxC]x3Cx5 fore of a/b1 domains (fig. 1). Other Bivalvia MTs (e.g., [CxC]x3[CxC]) (table 1 and supplementary fig. S2I, Crassostrea MTIIIs in the order Ostreoidea and PmarMT2 Supplementary Material online). The structure of the of Pinctada martensii in the order Pterioida) were organized Monoplacophora MT was therefore of b2/b1 domains (fig. 1). in two b2 domains. These MTs probably derived from an ancestral b2/b1 form (fig. 1). Deviations from these structures were found, ranging from loss or gain of some cysteines or Aculifera Clade small protein fragments (e.g., MquMT1 and CgiMTIV), to Polyplacophora. We identified five MTs in Polyplacophora significant structural modifications, such as those previously species: Acanthochitona crinita (AcriMT1), Chaetopleura api- described in a sphaeriid clam (Pisidium coreanum) PcorMT1 culata (CapMT1), Chiton olivaceus (ColMT1), Tonicella lineata 430 Modularity in Protein Evolution . doi:10.1093/molbev/msaa230 MBE (TliMT1), and Leptochiton rugatus (LruMT1) (supplementary MTs in many Mollusca species, which denoted an intricate fig. S2J and table S2, Supplementary Material online). and dynamic evolutionary history of mollusk MTs. Polyplacophora MTs were 70–73 amino acid long with 21 cysteines (28.8–30.0%) organized in a N-terminal 12-Cys a Discussion domain ([CxC]x5[CxC]x3Cx4[CxC]x3[CxC]x3[CxC]x2C) linked Functional Evolution of Mollusk MTs by three residues to a C-terminal 9-Cys b1 domain ([CxC]x3– Evolution of the Metal-Binding Capacity 4[CxC]x3Cx5[CxC]x3[CxC]) (table 1 and supplementary fig. S2J, Supplementary Material online). The structure of Most mollusk MTs are bimodular proteins with 18 (in b2–3/b1 Polyplacophora MTs was therefore of a/b1 domains (fig. 1). MTs), 19 (in c/b1), 21 (in a/b1), or 23 (in d/b1) cysteines. Neritimorpha b3/b1 MTs render homometallic complexes Solenogastres. We identified eight MTs of Solenogastres spe- with six divalent ions, either Zn(II) or Cd(II) (fig. 3), meaning cies: Alexandromenia crassa (AcraMT1 partial), that each 9-Cys domain is designed to allocate three divalent Amphimeniidae sp. (AspMT1 partial), Micromenia fodiens metal ions. This capacity is similar to those reported for other (MfoMT1 partial), Neomenia carinata (NcaMT1), Neomenia bimodular gastropod MTs (Perez-Rafael et al. 2012, 2014; megatrapezata (NmegMT1 and NmegMT2), and Palacios et al. 2014; Dvorak et al. 2018) and agrees with 3D Neomeniomorpha sp. (NspMT1 and NspMT2 partial) (sup- structural analysis of Littorina littorea (Baumann et al. 2017) plementary fig. S2K and table S2, Supplementary Material and Helix pomatia MTs (Beil et al. 2019). The metal-binding online). Full-length Solenogastres MTs (i.e., NcaMT1, capacity of Patellogastropoda c/b1 LgiMTs, both with 19 cys- NmegMT1, and NspMT1) were 65–66 amino acid long teines, is slightly higher since they bind seven divalent metal with 18–19 cysteines (27.3–29.3%) organized in an N-terminal ions (fig. 2A and B), similar to the 21-Cys a/b1 MTs of Bivalvia 9-Cys b2 domain ([CxC]x5[CxC]x3[CxC]x2–4[CxC]x3–4C) (Munoz et al. 2002; Orihuela et al. 2008). The successful syn- linked by six residues to a C-terminal 9-Cys b1 domain thesis of the c domain of LgiMT2, with ten Cys residues, and ([CxC]x3–4[CxC]x3Cx5[CxC]x3[CxC]) with an additional cys- the characterization of the species produced in Cd(II)- teine in NmegMT1 and NspMT1 sequences (table 1 and enriched media indicate that the LgiMTs render metal- supplementary fig. S2K, Supplementary Material online). aggregates containing seven Cd(II) ions because these MTs The structure of these MTs was therefore of b2/b1 domains are capable of binding three Cd(II) ions in their b1 domain, (fig. 1). Interestingly, Neomenia megatrapezata species had a whereas their c domain allocates four Cd(II) ions (fig. 2C). The second MT, NmegMT2, whose N-terminal domain was a 12- metal-binding capacity of MTs appears, therefore, to rely on Cys a domain ([CxC]x5[CxC]x3Cx5[CxC]x3[CxC]x3[CxC]x2C) the number and position of the cysteines in the different linked by seven residues to a C-terminal 9-Cys b1 domain domains and, as expected, the higher the cysteine content, ([CxC]x3[CxC]x3Cx5[CxC]x3[CxC]) (table 1 and supplemen- the higher the metal-binding capacity. tary fig. S2L, Supplementary Material online). The NmegMT2 In that sense, an effective evolutionary strategy for increas- structure was, therefore, of the a/b1 type, meaning that two ing the metal-binding capacity of MTs has been the design of structurally different MTs coexisted in the same Solenogastres multimodular forms with high cysteine content and a high species (fig. 1). capacity of metal binding (Niederwanger, Dvorak et al. 2017; Caudofoveata. We identified four MTs in Caudofoveata spe- Palacios et al. 2017; Calatayud et al. 2018). Multimodular MTs cies: Chaetoderma nitidulum (CniMT1), Falcidens caudatus had been identified in a few Bivalve and Gastropoda species (FcaMT1), Falcidens sagittiferus (FsaMT1), and Scutopus ven- (Tanguy and Moraga 2001; Jenny et al. 2004, 2016; Baumann trolineatus (SveMT1) (supplementary fig. S2M and table S2, et al. 2017; Nam and Kim 2017; Niederwanger, Calatayud et al. Supplementary Material online). The Caudofoveata MTs 2017; Palacios et al. 2017; Schmielau et al. 2019; Pedrini- were 73–78 amino acid long with 23 cysteines (29.5–31.5%) Martha et al. 2020), and our results have increased this list organized in a novel N-terminal 14-Cys domain with 16 new proteins containing a variable number of re- ([CxC]x3[CxC]CCx4Cx3–7[CxC]x3CCx4[CxC]C) that we peated domains. Sequence comparisons (fig. 4 and supple- named d domain, linked by three residues to a C-terminal mentary fig. S3, Supplementary Material online) and 9-Cys b1 domain ([CxC]x3–4[CxC]x3Cx5[CxC]x3[CxC]) (ta- structural analysis (Baumann et al. 2017) indicate that these ble 1 and supplementary fig. S2M, Supplementary Material multimodular MTs originated by N-terminal duplications of online). The structure of Caudofoveata MTs was therefore of the b3 domain, in agreement with the idea that proteins tend d/b1 domains (fig. 1). to increase in length mainly by the gain of sequences at the 50 - In summary, we have identified more than 270 MTs in 189 end of their genes (Toll-Riera and Alba 2013). The evolution different species distributed across the eight molluskan clas- of such multimodular MTs in some mollusk species suggests ses, 64% as single copy sequences (supplementary table S2, that diverse lineages have had to adapt to different conditions Supplementary Material online; the existence of additional of metal bioavailability and stress, despite the ecophysiolog- MTs in some species cannot be excluded because some se- ical determinants that have favored them in only some spe- quence databases are still in progress). Our data showed that cies remain, however, unknown. a single MT with a two-domain (i.e., bimodular) structure was the predominant type of MTs in most Mollusca species (fig. 1 Evolution of the Metal-Binding Preference and table 1). Our results revealed, however, many exceptions The evolution of MTs with different metal preferences has to this situation, with MT multiplicity and/or multimodular usually been associated with scenarios of MT multiplicity, in 431 Calatayud et al. . doi:10.1093/molbev/msaa230 MBE which neofunctionalization processes yielded MT duplicates diversification might be related to the extraordinary evolu- with new metal-binding selectivities. Sixty-seven species tionary success of the phylum, with species that have colo- patchily distributed across the mollusk phylogeny possess nized and adapted to very diverse habitats around the world. at least two MTs (supplementary table S2, Supplementary Material online), and their sequence (supplementary fig. S2, Structural Evolution of Mollusca MTs Supplementary Material online) and domain conservation Bimodular Structure of Mollusca MTs (fig. 1 and table 1) suggest that most of them originated after Vertebrate MTs have a bimodular structure made of two the splitting of the main Mollusk groups by lineage-specific independent functional domains—an 11–12-Cys a domain duplications. For instance, a gene duplication in the ancestor and a 9-Cys b domain—each one capable to bind metal ions of the Neritimorpha class resulted in two MTs that according (Braun et al. 1986; Capdevila et al. 1997; Cols et al. 1999). By to the analysis of N. peloronta NpeMT1 and NpeMT2 di- comparison with vertebrates, the bimodular structure has verged in their metal preferences (i.e., a Zn-thionein character been extended to gastropod (Palacios et al. 2011; Perez- for NpeMT1 and a Cd-thionein nature for NpeMT2; fig. 3). In Rafael et al. 2012, 2014; Dvorak et al. 2018; Beil et al. 2019) contrast, the duplication found in L. gigantea appears more and bivalve MTs (Jenny et al. 2004, 2016; Nam and Kim 2017; recent since both duplicates are 96% identical (supplemen- Yingprasertchai et al. 2019). Our results spread the bimodular tary fig. S1, Supplementary Material online) and still share a structure to the MTs of all molluskan classes since 90% of Cd binding preference (fig. 2). Our results support the idea their MTs are two-domain proteins. In these bimodular MTs, that metal-binding preference and hence, functional specific- an N-terminal domain that it is variable depending on the ity, does not mainly rely on the number and position of the taxon (an a domain in Bivalvia, Cephalopoda, and cysteines (>94% identical between NpeMT1 and NpeMT2) Polyplacophora MTs; a b2 domain in Scaphopoda, but on the nature of the noncoordinating amino acids (70% Monoplacophora, and Solenogastres MTs; a d domain in different between NpeMT1 and MpeMT2) distributed along Caudofoveata MTs; and a b3 domain in Gastropoda MTs, the protein sequence (Palacios et al. 2011; Perez-Rafael et al. with the exception of the c domain in Patellogastropoda 2014; Dallinger et al. 2020), although we are still far from being MTs) is linked to a conserved b1 domain at the C-terminal able to predict metal preference based on the analysis of the region (fig. 1 and table 1). The pervasiveness of the C-terminal noncoordinating amino acids. b1 domain suggests a conserved role for this domain, prob- We do not know the biological determinants that favored ably related to the stabilization of the 3D cluster structure of the evolution of MTs with different metal preferences in cer- the entire protein (Dallinger et al. 2020). tain mollusk lineages, but the colonization of new habitats (Dallinger et al. 2020) and the emergence of physiological novelties (Dallinger et al. 2005; Höckner et al. 2011) have Modular Evolution of MTs: Ancient and Recent Domains been proposed as significant evolutionary factors. We do The origin and the evolutionary relationships of the distinct a, not know either the binding selectivity of the ancestral mol- b1, b2, b3, c, and d domains were intriguing. The finding of a, lusk MT, but the widespread cadmium-binding capacity of b1, and b2 domains in diverse molluskan classes together many MTs—not only in mollusk but in diverse marine ani- with the presence of a/b1 and b2/b1 MTs in species of mals (Narula et al. 1995; Riek et al. 1999; Valls et al. 2001; both Conchifera and Aculifera (fig. 1) supports that the origin Guirola et al. 2012; Calatayud et al. 2018)—along with the of the domains predated the diversification of the phylum, ancient origin a and b domains (see below), lead us to spec- which has estimated at more than 545 Ma (Kocot et al. 2020), ulate that ancestral MTs might have been a detoxification and suggests that an ancient MT multiplicity was subse- system that was later co-opted for homeostatic functions for quently maintained or lost in a lineage-specific manner. In essential metals. Other scenarios are possible but since Cd is a addition, a possible connection between a and b domains highly toxic metal because it competes for Zn-dependent might be envisaged based on the number and configuration cellular processes, and Cd is frequently found with Zn in of the cysteine motifs (fig. 5). Thus, the three b domains ore deposits of the earth crust, an early evolution of Cd- might have derived from an ancestral a domain trimmed detoxifying MTs could have conferred a significant physiolog- at the C-terminal end (b1), at the middle (b2), and at the ical advantage to marine organisms. This advantage would be N-terminal end (b3), respectively. This possibility challenges especially important after increased Cd levels during the classical view that b domains represent ancestral forms, Paleozoic era (Dallinger et al. 2020), concomitantly with the and that a domains evolved later to provide detoxification Cambrian explosion and the emergence of most animal capacity in front of toxic metals such as cadmium (Cols et al. phyla. From the ancestral MT, different metal-selective MTs 1999). would have independently evolved in different molluskan In contrast to the ancient origin of a, b1, and b2 domains, lineages: Cu-selective MTs in Heterobranchia species the restricted distribution of b3, c, and d domains in some (Höckner et al. 2011; Perez-Rafael et al. 2011; Palacios et al. mollusk lineages suggests a more recent origin, probably con- 2014), Zn-selective forms in Neritimorpha gastropods (this comitant with the appearance of these taxonomic groups work) and bivalves (Orihuela et al. 2008), and metal- (fig. 1). Novel N-terminal domains would have replaced for- unselective MTs in Vetigastropoda and Heterobranchia line- mer and older ones specifically in some mollusk lineages: the ages (Höckner et al. 2011; Perez-Rafael et al. 2011, 2012, 2014; b3 domain in Gastropoda, the c domain in Niederwanger, Calatayud, et al. 2017). This functional Patellogastropoda, and the d domain in Caudofoveata. 432 Modularity in Protein Evolution . doi:10.1093/molbev/msaa230 MBE Other scenarios of domain evolution cannot be ruled out, but used as queries in TBlastN searches in expressed sequence they would require assuming complex processes of parallel or tags and genomic NCBI and eSnail (http://soft.bioinfo-min- convergent evolution to justify the current distribution of the zhao.org/esnail/index.html) databases. In addition, RNA-SRA different domains throughout Mollusca. for each mollusk species deposited in NCBI were Blast searched using as queries MT sequences from the nearest phylogenetically species as well as from different mollusk spe- De Novo Domain Evolution cies covering all the major clades. Raw sequence data were Whereas there might be an evolutionary relationship be- retrieved from the SRA and assembled using SeqMan 8.0.2 tween a and b domains, any link among these domains (Pro Assembler) software from the DNASTAR Lasergene and the novel c and d domains is obscured by their differ- package, and manually inspected in order to reconstruct ences. Although it cannot be ruled out that c and d domains new MT sequences. The MT nature of each new identified derived from a or b domains that have diverged too much for sequence was evaluated by BlastX searches against metazoan homology to be recognized, the sequence and cysteine motifs NCBI nonredundant protein sequence database. The amino in the c and d domains are so divergent from those in a and b acid sequences and the accession numbers of the retrieved domains that they seem to have evolved de novo. Recent MTs are provided in supplementary table S2, Supplementary analyses have shown that, indeed, de novo evolution is more Material online. frequent than previously thought (Neme and Tautz 2013; Toll-Riera and Alba 2013; Weisman and Eddy 2017; Levy 2019). Studies about the emergence of novel domains in hu- Characterization of L. gigantea MT Genes Collection of three L. gigantea specimens, dissection of their man proteins, for instance, have revealed more than 400 hepatopancreas, RNA extraction, and storage in RNA later “young” domains, 164 of which are found combined with (Thermo Fisher Scientific, Waltham, CA) were performed by older ones and preferentially located at the N-terminus of one of us (D.J.E.). Genomic DNA was obtained from hepato- the proteins (Toll-Riera and Alba 2013). These new domains pancreatic tissue disrupted with the TissueLyser II (Qiagen, are rich in low-complexity sequences (Toll-Riera et al. 2012) Hilden, Germany) procedure, and following the manufac- and tend to be structurally disordered (Moore and Bornberg- turer’s instructions of DNeasy Plant Mini Kit (Qiagen) for Bauer 2012). Such structural features match well those of DNA extraction. The DNA concentration, purity, and integ- MTs, which are considered as low complexity and intrinsically rity were checked using Tecan Infinite M200 (Tecan Group disordered proteins. Thus, de novo emergence of MT Ltd, Switzerland) measuring the absorbance at 260 and domains might be relatively easy under an evolutionary per- 280 nm. For the RNA extraction, tissue of the midgut gland spective because the only requirement for a peptide to func- was homogenized with glass beads using the Precellys ho- tion as a metal ion chelator would be a high content of mogenizer (Bertin Instruments, Montigny-le-Bretonneux, coordinating residues (e.g., cysteines) and a relative small France). Total RNA was isolated applying the RNeasy Plant length that favored the polypeptide folding (Capdevila and Mini Kit (Qiagen) including on-column DNase I digestion Atrian 2011). De novo evolution of MTs has been, indeed, (Qiagen) according to manufacturer’s constructions. RNA in- implicitly stated from diverse evolutionary studies concluding tegrity was checked by visualization on a 1.5% agarose gel that MTs likely evolved more than once in different animal (Biozym, Hessisch Oldendorf, Germany). For cDNA synthesis, phyla (Capdevila and Atrian 2011; Blindauer 2014; Isani and 450 ng total RNA was used applying the RevertAid Reverse Carpene 2014; Ziller and Fraissinet-Tachet 2018). Transcriptase (Fermentas by Thermo Fisher Scientific). In summary, the evolution of the mollusk MTs is intriguing. The genomic region containing the putative MT genes of At the short term (at low taxonomic ranks), it appears to L. gigantea was PCR amplified, cloned PCR primers (supple- have followed the habitual evolutionary patterns based on mentary table S3, Supplementary Material online) were progressive accumulation of changes in the sequence, and on designed based on the L. gigantea genome project (Scaffold duplications or losses of internal domains or genes. At the and resequenced. 35, NW_008709190.1) in order to amplify long term (at high taxonomic ranks), in contrast, MT evolu- overlapping fragments of different sizes (from 500 bp to 4 kb), tion seems to have been mainly impacted by emergence of covering the entire putative MT-containing genomic region. new structural domains. The modular structure of mollusk For each PCR reaction, 1 ng of genomic DNA was amplified MTs makes the analyses of their domain organization and using selected pairs of primers and the Phusion High-Fidelity cysteine motifs more informative for inferring their evolution DNA Polymerase (Invitrogen, Thermo Fisher Scientific) in a during the diversification of the phylum than the classic final 25 ll reaction. PCR conditions were 98  C 30 s (s); 35 comparisons of sequences, which may be biased toward a cycles of 98  C 10 s, 58  C 30 s, and 72  C 3 min; and 72  C general cysteine-richness due to the structural and functional 10 min. PCR products were visualized in 0.7% agarose gels, requirements of metal binding. isolated with the GelElute Plasmid Miniprep Kit (Sigma- Aldrich, Merck KGaA, Darmstadt, Germany), and cloned Materials and Methods with TOPO TA Cloning Kit (Invitrogen, Thermo Fisher Database Searches and MT Identification Scientific). Plasmid DNA was purified from bacteria using Molluskan MT sequences were identified from public data- the GeneElutet Plasmid Miniprep Kit (Sigma-Aldrich), bases by Entrez searches using “metallothionein” and screened for insert presence by digestion with EcoRI (EcoRI “Mollusca” as queries. Retrieved MT sequences were then Fast Digest Restriction Enzyme, Invitrogen, Thermo Fisher 433 Calatayud et al. . doi:10.1093/molbev/msaa230 MBE Scientific), and sequenced at the Scientific and Technological overnight at 17  C, thus enabling separation of the metal- Centers of the University of Barcelona using the Big Dye MT complexes from the GST that remained bound to the Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) sepharose matrix. The eluted metal-MT complexes were con- in an automatic sequencer (ABIPRISM 310, Applied centrated with a 3-kDa Centripep Low Concentrator Biosystems). Several internal primers (supplementary table (Amicon, Merck) and fractionated on a Superdex-75 FPLC S3, Supplementary Material online) were used to fully se- column (GE Healthcare) equilibrated with 20 mM Tris–HCl, quence the overlapping PCR fragments, which were manually pH 7.0. The protein-containing fractions, identified by their assembled in order to reconstruct the entire genomic region. absorbance at 254 nm, were pooled and stored at 80  C The prediction of the L. gigantea MT genes was corrobo- until use. rated by the PCR amplification of the corresponding cDNAs using gene specific primers (supplementary table S3, Supplementary Material online) designed with CLC main Analysis of Metal-MT Complexes workbench (Version 6.9), and Advantage 2 polymerase Protein quantification and element composition of all the (Clontech, Takara Bio Europe, Saint-Germain-en-Laye, samples were achieved by ICP-AES measurements performed France) in a final 50 ll reaction. Cycling conditions were in a Optima 4300DV (Perkin-Elmer, MA, USA) apparatus (S, 95  C 1 min; 30 cycles 95  C 30 s, 53  C (for LgiMT2)/55.5  C 182.040 nm; Zn, 213.856 nm; Cd, 228.802 nm; and Cu, (for LgiMT1) 30 s, and 68  C 40 s; and 68  C 5 min. PCR prod- 324.803 nm) under conventional conditions following an al- ucts were visualized on a 1.5% agarose gels, purified using the ready established method (Bongers et al. 1988). QIAquick gel extraction kit (Qiagen) and cloned with the Molecular weights were determined by ESI-MS, in a TOPO TA Cloning Kit (Invitrogen, Thermo Fisher MicroTof-Q instrument (Bruker Daltonics Gmbh, Bremen, Scientific). Insert containing plasmids were isolated using Germany) connected to a Series 1100 HPLC pump (Agilent the QIAprep Spin Miniprep Kit (Qiagen) and sent for se- Technologies) controlled by the Compass Software. The in- quencing to Microsynth (Balgach, Switzerland). Sequences strument was calibrated with ESI-L Low Concentration were analyzed via CLC main workbench (Version 6.9). Turning Mix (Agilent Technologies, Santa Clara, CA). Metallated forms were detected under native conditions: Production and Purification of Recombinant of Metal- 20 ml of sample injected through a PEEK tube at 30– MT Complexes 50 ml min1 in a 3.5–5.0-kV capillary-counter voltage, at 90– Synthetic cDNAs codifying the selected MTs (i.e., LgiMT1, 110  C of desolvation temperature, and with dry gas at LgiMT2, LgiMT2-c domain, NpeMT1, and NpeMT2) were 6 l min1. Spectra were recorded between a m/z range from provided by Synbiotech (Monmouth Junction, NJ, USA) 800 to 3,000. The liquid carrier was a 90:10 mixture of 15 mM cloned in the pGEX-4T-1 expression vector (GE Healthcare). ammonium acetate and acetonitrile at pH 7.0. All molecular Recombinant plasmids were transformed in E. coli BL21 strain, masses were calculated according to the bibliography (Fabris a protease-deficient strain used for heterologous protein ex- et al. 1996). pression. For heterologous protein production, 500 ml of Luria-Bertani (LB) medium with 100 lg/ml ampicillin was inoculated with E. coli BL21 cells transformed with the corre- Supplementary Material sponding recombinant plasmid. After overnight growth at 37  C/250 rpm, the cultures were used to inoculate 5 l of fresh Supplementary data are available at Molecular Biology and LB-100 lg/ml ampicillin medium. Gene expression was in- Evolution online. duced with 100 lM isopropyl-b-D-thiogalactopyranoside for 3 h. After the first 30 min of induction, cultures were supple- mented with ZnCl2 (300 lM), CdCl2 (300 lM), or CuSO4 Acknowledgments (500 lM) in order to generate metal-MT complexes. Cells Parts of this study were financed by a cooperation grant to were harvested by centrifugation for 5 min at 9,100  g R.D. from the Austrian Science Fund (DACH grant No I 1482- (7,700 rpm), and bacterial pellets were suspended in 125 ml N28). Researchers from Barcelona want to acknowledge the of ice-cold phosphate-buffered saline (PBS) (1.4 M NaCl, Spanish Ministerio de Ciencia e Innovacion and FEDER for the 27 mM KCl, 101 mM Na2HPO4, 18 mM KH2PO4, and 0.5% projects BIO2015-67358-C2-2-P (M.C. and O.P.) and BIO2015- v/v b-mercaptoethanol). Resuspended cells were sonicated 67358-C2-1-P (R.A.). M.C. and O.P. are members of the “Grup (Sonifier Ultrasonic Cell Disruptor) 8 min at voltage 6 with de Recerca de la Generalitat de Catalunya,” ref. 2017SGR-864, pulses of 0.6 s, and then centrifuged for 40 min at 17,200  g and R.A. of ref. 2017SGR-1665. M.G.-R. acknowledges to the (12,000 rpm) and 4  C. Soluble protein extracts containing UAB the PIF grant. D.J.E. acknowledges support from the GST-MT fusion proteins were incubated with glutathione United States National Science Foundation (Grant No. DEB- sepharose beads (GE Healthcare) for 1 h at room temperature 1355230). We also thank the Servei d’Analisi Quımica (SAQ) with gentle rotation. GST-MT fusion proteins bound to the at the Universitat Autonoma de Barcelona (ICP-AES, ESI-MS) sepharose beads were washed with 30 ml of cold 1 PBS for allocating instrument time, the Centres Cientıfics i bubbled with argon to prevent oxidation. After three washes, Tecnologics (CCiT) at the Universitat de Barcelona for DNA GST-MT fusion proteins were digested with thrombin (GE sequencing, and to Sebastian Artime for the experimental Healthcare, 25 U/l of culture or SERVA, 25 U/l of culture) support. 434 Modularity in Protein Evolution . doi:10.1093/molbev/msaa230 MBE Data Availability Dallinger R, Chabicovsky M, Hödl E, Prem C, Hunziker P, Manzl C. 2005. Copper in Helix pomatia (Gastropoda) is regulated by one single cell The data underlying this article are available in NCBI (https:// type: differently responsive metal pools in rhogocytes. 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