## Literature Cited

Aizenberg J, Lambert G, Weiner S, Addadi L. 2002. Factors involved in the formation of amorphous and crystalline calcium carbonate: a study of an ascidian skeleton. J Am Chem Soc. 124(1):32–39.

Altermann W, Kazmierczak J, Oren A, Wright DT. 2006. Cyanobacterial calcification and its rock-building potential during 3.5 billion years of Earth history. Geobiology 4(3):147–166.

Altschul SF, et al. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25(17):3389–3402.

Benzerara K, et al. 2014. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria. Proc Natl Acad Sci U S A. 111(30):10933–10938.

Bitard-Feildel T, Lamiable A, Mornon J-P, Callebaut I. 2018. Order in disorder as observed by the “hydrophobic cluster analysis” of protein sequences. Proteomics 18(21–22):e1800054.

Blondeau M, Benzerara, K, et al. 2018. Impact of the cyanobacterium *Gloeomargarita lithophora* on the geochemical cycles of Sr and Ba. Chem Geol. 483:88–97.

Blondeau M, Sachse M, et al. 2018. Amorphous calcium carbonate granules form within an intracellular compartment in calcifying cyanobacteria. Front Microbiol. 9:1768.

Blue CR, et al. 2017. Chemical and physical controls on the transformation of amorphous calcium carbonate into crystalline CaCO₃ polymorphs. Geochim Cosmochim Acta. 196:179–196.

Bradley JA, et al. 2017. Carbonate-rich dendrolitic cones: insights into a modern analog for incipient microbialite formation, Little Hot Creek, Long Valley Caldera, California. NPJ Biofilms Microbiomes. 3:32.

Bull PC, Cox DW. 1994. Wilson disease and Menkes disease: new handles on heavy-metal transport. Trends Genet. 10(7):246–252.

Callebaut I, et al. 1997. Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives. Cell Mol Life Sci. 53(8):621–645.

Cam N, et al. 2016. Selective uptake of alkaline earth metals by cyanobacteria forming intracellular carbonates. Environ Sci Technol. 50(21):11654–11662.

Cam N, et al. 2018. Cyanobacterial formation of intracellular Ca-carbonates in undersaturated solutions. Geobiology 16(1):49–61.

Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. 2009. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25(15):1972–1973.

Couradeau E, Benzerara K, Gerard E, Moreira D, Bernard S, Brown GE, Lopez-Garcia P. 2012. An early-branching microbialite cyanobacterium forms intracellular carbonates. Science 336:459–462.

De la Concepcion JC, et al. 2018. Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen. Nat Plants. 4(8):576–585.

De Wever A, et al. 2019. Evidence of high Ca uptake by cyanobacteria forming intracellular CaCO₃ and impact on their growth. Geobiology 17(6):676–690.

Eddy SR. 2011. Accelerated profile HMM searches. PLoS Comput Biol. 7(10):e1002195.

Finney AR, Innocenti Malini R, Freeman CL, Harding JH. 2020. Amino acid and oligopeptide effects on calcium carbonate solutions. Cryst Growth Des. 20(5):3077–3092.

Frangeul L, et al. 2008. Highly plastic genome of *Microcystis aeruginosa* PCC 7806, a ubiquitous toxic freshwater cyanobacterium. BMC Genomics. 9(274):274.

Gillespie JJ, et al. 2011. PATRIC: the comprehensive bacterial bioinformatics resource with a focus on human pathogenic species. Infect Immun. 79(11):4286–4298.

Gouy M, Guindon S, Gascuel O. 2010. SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 27(2):221–224.

Humbert J-F, et al. 2013. A tribute to disorder in the genome of the bloom-forming freshwater cyanobacterium *Microcystis aeruginosa*. PLoS One. 8(8):e70747.

Hyatt D, et al. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 11:119.

Jiang D, et al. 2013. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. Proc Natl Acad Sci U S A. 110(36):14664–14669.

Jiang D, et al. 2014. Atomic resolution structure of the *E. coli* YajR transporter YAM domain. Biochem Biophys Res Commun. 450(2):929–935.

Jumper J, et al. 2021. Highly accurate protein structure prediction with AlphaFold. Nature 596(7873):583–589.

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 30(4):772–780.

Kim S, et al. 2005. Transmembrane glycine zippers: physiological and pathological roles in membrane proteins. Proc Natl Acad Sci U S A. 102(40):14278–14283.

Komarek J, Johansen JR, Smarda J, Strunecky O. 2020. Phylogeny and taxonomy of Synechococcus-like cyanobacteria. Fottea 20(2):171–191.

Kuehlbrandt W. 2019. Structure and mechanisms of F-type ATP synthases. Annu Rev Biochem. 88: 515–549.

Lamiable A, et al. 2019. A topology-based investigation of protein interaction sites using hydrophobic cluster analysis. Biochimie 167:68–80.

Latour D, Salençon M-J, Reyss J-L, Giraudet H. 2007. Sedimentary imprint of *Microcystis aeruginosa* (cyanobacteria) blooms in grangent reservoir (Loire, France). J Phycol. 43(3):417–425.

Lefevre CT, Bazylinski DA. 2013. Ecology, diversity, and evolution of magnetotactic bacteria. Microbiol Mol Biol Rev. 77(3):497–526.

Leonov H, Arkin IT. 2005. A periodicity analysis of transmembrane helices. Bioinformatics 21(11):2604–2610.

Li J, et al. 2016. Biomineralization patterns of intracellular carbonatogenesis in cyanobacteria: molecular hypotheses. Minerals 6(1):10.

Li L, Stoeckert CJ, Roos DS. 2003. OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res. 13(9):2178–2189.

Marchler-Bauer A, Bryant SH. 2004. CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 32(Web Server Issue):W327–W331.

Marron AO, et al. 2016. The evolution of silicon transport in eukaryotes. Mol Biol Evol. 33(12):3226–3248.

Mehta N, Benzerara K, Kocar BD, Chapon V. 2019. Sequestration of radionuclides radium-226 and strontium-90 by cyanobacteria forming intracellular calcium carbonates. Environ Sci Technol. 53(21):12639–12647.

Mermet-Bouvier P, Chauvat F. 1994. A conditional expression vector for the cyanobacteria *Synechocystis* sp. strains PCC6803 and PCC6714 or *Synechococcus* sp. strains PCC7942 and PCC6301. Curr Microbiol. 28(3):145–148.

Mirdita M, et al. 2021. ColabFold—making protein folding accessible to all. bioRxiv. doi:10.1101/2021.08.15.456425.

Monteil CL, et al. 2021. Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria. ISME J. 15(1):1–18.

Moreira D, et al. 2017. Description of *Gloeomargarita lithophora* gen. nov., sp nov., a thylakoid-bearing, basal-branching cyanobacterium with intracellular carbonates, and proposal for *Gloeomargaritales* ord. nov. Int J Syst Evol Microbiol. 67(3):653–658.

Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 32(1):268–274.

Pettersen EF, et al. 2004. UCSF chimera—a visualization system for exploratory research and analysis. J Comput Chem. 25(13):1605–1612.
Ragon M, Benzerara K, Moreira D, Tavera R, López-García P. 2014. 16S rDNA-based analysis reveals cosmopolitan occurrence but limited diversity of two cyanobacterial lineages with contrasted patterns of intracellular carbonate mineralization. Front Microbiol. 5:331.

Reynolds CS, Rogers DA. 1976. Seasonal variations in the vertical distribution and buoyancy of *Microcystis aeruginosa* Kütz. emend. Elenkin in Rostherne Mere, England. Hydrobiologia 48(1):17-23.

Riding R. 2012. A hard life for cyanobacteria. Science 336(6080):427-428.

Robert X, Gouet P. 2014. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42(Web Server Issue):W320-W324.

Senes A, Engel DE, DeGrado WF. 2004. Folding of helical membrane proteins: the role of polar, GxxxG-like and proline motifs. Curr Opin Struct Biol. 14(4):465-479.

Shimodaira H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst Biol. 51(3):492-508.

Veaudor T, et al. 2018. Overproduction of the cyanobacterial hydrogenase and selection of a mutant thriving on urea, as a possible step towards

the future production of hydrogen coupled with water treatment. PLoS One. 13(6):e0198836.

Wang X, et al. 2021. The evolution of calcification in reef-building corals. Mol Biol Evol. 38:3543-3555.

Webb B, Sali A. 2016. Comparative protein structure modeling using MODELLER. Curr Protoc Protein Sci. 86:2.9.1-2.9.37.

Weiner S, Dove PM. 2003. An overview of biomineralization processes and the problem of the vital effect. Rev Minerol Biochem. 54: 1-29.

Yang M, Derbyshire MK, Yamashita RA, Marchler-Bauer A. 2020. NCBI's conserved domain database and tools for protein domain analysis. Curr Protoc Bioinformatics. 69(1):e90.

Yarra T, Blaxter M, Clark MS. 2021. A Bivalve Biomineralization Toolbox. Mol Biol Evol. 38(9):4043-4055.

Zimmermann L, et al. 2018. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J Mol Biol. 430(15):2237-2243.

Associate editor: Tal Dagan
AlphaFold2-aided description of CoBaHMA, a novel family of
bacterial domains within the Heavy-Metal Associated superfamily

Geoffroy Gaschignardª, Maxime Milletª, Apolline Bruley, Karim Benzerara, Manuela Dezi,

Feriel Skouri-Panet, Elodie Dupratᵇ, Isabelle Callebautᵇ

Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590, Institut de
Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005 Paris, France

a. Geoffroy Gaschignard and Maxime Millet should be considered joint first authors.

b. Elodie Duprat and Isabelle Callebaut should be considered joint senior authors

Correspondence :

Elodie Duprat and Isabelle Callebaut, Sorbonne Université, Muséum National d'Histoire Naturelle, UMR CNRS 7590, Institut de Minéralogie, de Physique des Matériaux et de Cosmochimie, IMPMC, 75005 Paris, France. Email: elodie.duprat@sorbonne-universite.fr
(E.D.) and isabelle.callebaut@sorbonne-universite.fr (I.C.).
**ABSTRACT**

Three-dimensional structure information, now available at the proteome scale, may facilitate the detection of remote evolutionary relationships in protein superfamilies. Here, we illustrate this with the identification of a novel family of protein domains related to the ferredoxin-like superfold, by combining (i) transitive sequence similarity searches, (ii) clustering approaches and (iii) the use of AlphaFold2 3D structure models. Domains of this family called CoBaHMA, were initially identified in relation with the intracellular biomineralization of calcium carbonates by Cyanobacteria. They are part of the large Heavy-Metal Associated (HMA) superfamily, departing from the latter by specific sequence and structural features. In particular, most CoBaHMA domains share conserved basic amino acids, forming a positively charged surface which is likely to interact with anionic partners. CoBaHMA domains are found in diverse modular organizations in bacteria, existing in the form of monodomain proteins or as part of larger proteins, some of which are made up of membrane domains involved in transport or lipid metabolism. This suggests that the CoBaHMA domains may exert a regulatory function, involving interactions with anionic lipids. This hypothesis might have a particular resonance in the context of the compartmentalization observed for Cyanobacteria intracellular calcium carbonates.

**KEYWORDS:** Heavy-Metal Associated, AlphaFold2, sequence similarity search, graph clustering, modular organization, functional annotation, P1B-type ATPase, ABC transporter, PAP2, biomineralization, CoBaHMA

**ABBREVIATIONS**

aa: amino acids, A: Actuator, ABC: ATP-Binding Cassette, AF2: AlphaFold2, AFDB: AlphaFold Protein Structure DataBase, ATPDB: ATP-Binding Domain, ccya: calcyanin, CoBaHMA: Conserved Basic residues HMA, DAG: diacylglycerol, GlyZip: glycine-zipper, HCA: Hydrophobic Cluster Analysis, HMA: Heavy Metal-Associated, MSA: Multiple Sequence Alignment, N: Nucleotide, NBD: Nucleotide-Binding Domain, P: phosphorylation, PAE: Predicted Aligned Error, PAP2: type 2 phosphatidylglycerol phosphatase, PDB: Protein Data Bank, PG: phosphatidylglycerol, pLDDT: predicted Local Distance Difference Test, R: regulatory, RSSs: Regular Secondary Structures, S: auxiliary membrane, SCOP: Structural Classification of Proteins, TM: Transmembrane, TMD: Transmembrane Domain
55

56

FUNDING INFORMATION

57 This work was supported by the Agence Nationale de la Recherche (ANR Harley, ANR-19-

58

CE44-0017-01; ANR PHOSTORE, ANR- 19-CE01-0005).

59
# 1. Introduction

Superfolds are folds observed in a large number of evolutionary unrelated protein domain superfamilies ¹ and are characterized by compact super-secondary structure patterns ²,³. One of these superfolds is the ferredoxin-like fold, found in 62 superfamilies according to the SCOPe classification (d.58, ⁴) and present in many domains with various functions ⁵,⁶. It is made of a repeated β-α-β super-secondary structure, forming a four-stranded β-sheet, with the two α-helices packed into an α-β sandwich. As for other superfolds, the ferredoxin-like fold is subject to circular permutations, a mechanism which allows adaptation and the emergence of new functions. This can be visualized by the ligation of the amino- and carboxyl-termini and subsequent cleavage at another site ⁷,⁸. Among the 62 superfamilies comprising the ferredoxin-like fold, the Heavy-Metal Associated (HMA) superfamily (SCOP d58.17) contains the eponymous HMA family (Figure 1A). Domains of this HMA family contain two conserved cysteine residues involved in heavy metal binding. They are found in a variety of metal-trafficking proteins, which play essential roles in transport and homeostasis ⁹,¹⁰. While they can be found alone, HMA domains are also part of diverse domain architectures, especially associated with P<sub>IB</sub>-type ATPases, which are integral membrane proteins allowing transport of metals across cell membranes ¹¹.

We recently identified a novel family of domains belonging to the HMA superfamily, called CoBaHMA (after *Conserved Basic residues HMA*). This discovery originates from the characterization of a novel family of two-domain proteins, named calcyanin, which is associated with intracellular biomineralization of calcium carbonates by cyanobacteria ¹². Calcyanins share a common architecture consisting in a conserved C-terminal domain, made of a three-fold repeated, unusually very long glycine zipper motif ((GlyZip)₃). Glycine zippers themselves consist of repeated GXXXG motifs, commonly found in transmembrane domains and bacterial toxins ¹³,¹⁴. The calcyanin (GlyZip)₃ domain is preceded by a variable N-terminal domain, specific to the 4 distinct calcyanin subgroups identified to this date, which are found in distinct clades of cyanobacteria. The N-terminal domain of one of these calcyanin subgroups is a CoBaHMA domain. It is represented by 15 sequences described in ¹². It shares significant sequence similarities with HMA domains, as well as plant integrated HMA domains ¹⁵ and YAM domains found in the C-terminal part of the bacterial YajR, an integral membrane protein
which belongs to the Major Facilitator Superfamily (MFS, ¹⁶,¹⁷). These three families of domains share the same 3D structure, but only the first one (HMA) is referenced in the SCOPe classification (d.58.17.1, ⁴). Sequence analysis and molecular modeling ¹² have revealed specific features of the CoBaHMA family, including the presence of an additional strand β0 at the N-terminus of the domain, which replaces the C-terminal strand β4, thus conforming to the circular permutation scenario described before (Figure 1B). Calcyanins' CoBaHMA domains are also characterized by the presence of conserved basic amino acids in β0 (H) and β1 (R/K) strands, forming a charged patch on one side of the β-sheet. Despite the clear identification of all these specific sequence and structural features, the function(s) of CoBaHMA domains remain(s) unknown.

Here, we searched for the presence of CoBaHMA domains in proteins distinct from calcyanins. We posit that the identity of co-occurring domains or combinations of domains in other proteins may inform about their functional context ¹⁸⁻²⁰. For this purpose, we propose a novel methodological framework, taking advantage of the structural information provided by the Alphafold2 3D structure models ²¹ of the retrieved sequences, now widely available in the Alphafold DataBase ²². This allows a both sensitive and specific detection of CoBaHMA domains within the HMA superfamily. As a result, we describe a wide diversity of modular organizations in which CoBaHMA domains are included, with some yet-to-be-characterized regions. Moreover, the CoBaHMA domain appears to be specific to bacteria and frequently found associated with transmembrane domains involved in transport of substrates and lipid metabolism, suggesting a regulatory role with regard to these functions, probably via an interaction with charged lipids.

113
## 2. MATERIAL AND METHODS

The workflow followed by this study is shown in **Figure 2**.

### 2.1. Structure-guided detection of CoBaHMA domain sequences (Figure 2-A)

We searched the UniClust30/UniRef30 databases (version 2022_02, downloaded from https://gwdu111.gwdg.de/~compbiol/uniclust/2022_02/)²³ using the standalone version of HHBlits²⁴. The 15 sequences of CoBaHMA domains identified by Benzerara et al.¹² were used as individual probes. The sequence similarity search was made of 8 iterations with 50% hit coverage and an E-value threshold of 1e-3 for each sequence probe. For each iteration and each probe, we gathered the sequences extracted from the output a3m multiple sequence alignment. We removed trivial redundancy using an in-house python3 script, by merging entries with identical identifiers and whose sequences were identical on at least 50% of their respective length. In order to find which entry had a 3D model available in AlphaFold DB (https://alphafold.ebi.ac.uk/)²², we downloaded the accession_ids.csv file from http://ftp.ebi.ac.uk/pub/databases/alphafold/. Based on this file, we downloaded the 3D models corresponding to our entries from AlphaFold DB V4. We cropped each 3D model based on the boundaries of the CoBaHMA domain candidates identified with the HHBlits search. From the 3D coordinates of these sub-3D models, we computed the secondary structures associated with each amino acid using DSSP²⁵,²⁶. The 8 states output of DSSP was converted to 3 states using the EVA convention²⁷. Following this convention, α-helix, $\beta_{10}$ helix and π-helix are converted to helices (H), extended and isolated β-bridge to extended (E) and turn, bend and other to coils (C). To validate the presence of a secondary structure, we set a threshold of at least 3 consecutive amino acids with the same secondary structure assignment. As a result, we could infer the string of regular secondary structures of the sub-model. We set 4 criteria to identify a CoBaHMA domain: First, it should contain the secondary pattern EEHEEH(E) typical of CoBaHMA domains¹². Second, it should contain neither the Cys-X-X-Cys pattern, typical of HMA domains¹⁰, nor the Cys-X-X-X-Cys pattern typical of TRASH domains²⁸. Third, the secondary pattern should not start with EH, as this was characteristic of some false positives. Fourth, the first two beta strands had to be separated by a coil of 2 amino-acids or less, to avoid confusing a β0 strand of a CoBaHMA domain with the β4 strand of an
146

HMA domain, which is followed by a second HMA domain starting with a β1 strand). A final manual check based on the alignment of sequences was conducted to remove the remaining false positives. Entries meeting these 4 criteria and passing the manual check were considered as CoBaHMA domains.

147

148

149

150

We performed transitive searches with newly identified CoBaHMA domains. This process was iterated 6 times. We removed the redundancies out of the 6 iteration outputs by merging the entries that had the same identifiers and shared at least 50% of their sequences.

151

152

153

Finally, since HHblits alignment only gave the portions of the sequences that match the probes, we downloaded from UniProt (https://www.uniprot.org/)²⁹ the full sequences of each identifier that had at least one CoBaHMA domain in its sequence.

154

155

156

157

## 2.2. Annotation of the full-length sequences (Figure 2-B)

158

The TaxID and organism name associated with the sequences were retrieved using the UniProt REST API by downloading uniprot ttl files and serializing sequences information. Taxonkit³⁰ was used to convert TaxID into taxonomic lineages based on the NCBI taxonomy. Sequence functional features were assessed using several annotation tools. First, sequences were annotated with InterProscan³¹ and InterPro³² (interproscan-5.59-91.0). Additionally, domainMapper³³ [version 3.0.2] and the ECOD classification of known 3D structures of domains were used to complete the sequence annotation. This HMM parsing algorithm provides the detection of non-contiguous, insertional, and circularly permuted domains as well. Results from both tools were filtered with an E-value threshold of 1e-10. Transmembrane regions were annotated using deepTMHMM³⁴ [web version 1.0.20]. Finally, calcyanin sequences were detected using pCALF (see below and **Supplementary Data 1**). Hydrophobic Cluster Analysis (HCA³⁵,³⁶) was also used to assess the foldability of the analyzed sequences³⁷,³⁸.

159

160

161

162

163

164

165

166

167

168

169

170

171

172

## 2.3. Calcyanin detection and classification (Supplementary Data 1)

173

Sequences of calcyanins were detected in our data set using a dedicated tool called pCALF (standing for python CALcyanin Finder). pCALF used four Hidden Markov Model (HMM) profiles describing the glycine zipper triplication specific of calcyanin, called the (GlyZip)₃ motif, as described in ¹². The (GlyZip)₃ HMM profile describes the whole triplication while Gly1, Gly2 and Gly3 HMMs describe each glycine zipper individually. These HMMs profiles were

174

175

176

177
178

searched against amino acid sequences using pyHMMER ³⁹,⁴⁰. Additionally, a set of domains (Y-type, X-type, Z-type and CoBaHMA), known to be found together with the glycine zipper triplication, was used to annotate the N-terminal extremity of sequences. A sequence is classified as calcyanin if it has a significative hit against the (GlyZip)3 HMM profile (coverage threshold: 0.6 ; e-value threshold: 1e-20) and significative hits against Gly1, Gly2 and Gly3 zippers in this particular order (coverage threshold: 0.7, e-value threshold: 1e-10). A sequence is also classified as calcyanin if the second glycine zipper is missing and the N-terminal domain is of Y-type.

179

180

181

182

183

184

185

186

187

## 2.4. Classification of the full-length sequences (Figure 2-C)

188

Full length proteins were clustered using mmseqs2 and a coverage threshold of 0.97 ⁴¹ (coverage mode: 0, cluster mode: 0, identity threshold: 0%).

189

190

This threshold was the lowest that kept the length difference between the shortest and the longest sequences in a cluster below 50 residues and ensured that sequences belonging to the same cluster had the same number of domains capabilities. Sequences representative of each cluster, including singletons, were extracted and a self-versus-self search was performed using mmseqs2 (alignment mode: 0). Alignment results of the clustering and search were combined and filtered using a reciprocal coverage threshold of >0.7, an e-value threshold of <1e-10 and a sequence length amplitude lower than 50 residues. The bitscore was normalized by the length of the shortest sequences involved in the alignment ⁴² and an additional filter was applied using the median of normalized bitscore values (0.44).

191

192

193

194

195

196

197

198

199

200

A network was built using networkX ⁴³ with sequences as nodes and alignments as edges, weighted by the normalized bitscore. The network was refined iteratively in two steps: community detection and edges removal. The best partition was found using the Louvain Community Detection Algorithm ⁴⁴. For each community, edges were removed if one or both of its nodes has a length (i.e. number of residues) outside the interval [Xc – SEMc + N; Xc + SEMc – N], where Xc is the mean sequence length of a community, SEMc is the standard error of the mean sequence length of a community and N is a fixed number of residues N=35. Louvain community detection and edges removal were repeated until there was no more edge to remove and when the maximum length difference within all communities was less than 50 residues. Finally, the partitioning quality of the final network was assessed. Network layout

201

202

203

204

205

206

207

208

209
210

(edge-rendering and weighted spring embedded layout) and rendering were produced with
cytoscape ⁴⁵.

211

212

213

**2.5. Multiple alignment of representative sequences of the CoBaHMA family**

214

In order to build an alignment that is representative of CoBaHMA's diversity, and avoid
the over-representation of a subgroup of CoBaHMA domains, the 2305 CoBaHMA sequences
were clustered with mmseq2 (coverage mode: 0, cluster mode: 0, identity threshold: 60%,
coverage: 80%) ⁴¹. The representatives of the clusters and singletons were gathered,
amounting to a total of 1434 CoBaHMA sequences. To facilitate their alignment, the
sequences were split in subgroups of ~ 200 sequences. The sequences of these subgroups
were aligned using mafft v7.487 ⁴⁶ (maxiterate 1000; localpair), with some manual correction,
before merging them (mafft option: merge). The multiple sequence alignment (MSA) was
viewed and analyzed with Jalview 2.11.2.6 ⁴⁷.

215

216

217

218

219

220

221

222

223

WebLogo v2.8.2 ⁴⁸ was used on the Berkeley server (https://weblogo.berkeley.edu/logo.cgi)
to build the logo of the MSA, restricted to the CoBaHMA β strands. For the sake of clarity, we
removed the indels from alignment before making the logo.

224

225

226

227

**2.6. Analysis of the 3D structure models**

228

Manipulation and visualization of AF2 3D structure models were achieved using Chimera
⁴⁹. Based on ⁵⁰, the pLDDT were split in 4 categories: pLDDT ≥ 90 (very high confidence); pLDDT
∈ [70, 90[ (high confidence); pLDDT ∈ [50, 70[ (low confidence); and pLDDT <50 (non-
interpretable). The associated predicted aligned error (PAE), extracted from AFDB ²³ was also
considered in order to evaluate interdomain contacts. Each position of the PAE is the
incertitude in Å for the relative positions of 2 amino acids, whose positions in the sequence
are given by the x and y coordinates.

229

230

231

232

233

234

235

Structural similarities were searched using Foldseek ⁵¹. Three dimensional structure models
were analyzed in light of the multiple sequence alignments of the sequences of each
communities, built using mafft v7.487 ⁴⁶ and rendered using ESPript3 ⁵².

236

237

238

239
## 3. RESULTS

### 3.1. Identification of CoBaHMA domains not contained in calcyanins

We used the sequences of CoBaHMA domains from the 15 calcyanins reported in ¹² as queries and performed iterative, profile-based sequence similarity searches, combined with the consideration of structural features provided by AlphaFold2 models to specifically identify CoBaHMA domains in the HMA superfamily (see Material and Methods; **Figure 2**). The structural features specifically improved the discrimination between HMA and CoBaHMA domains, which are otherwise close sequence-wise. However, this specificity was achieved at the cost of a lower recovery of CoBaHMA domains, since not every sequence in the UniClust30 database had a model available in the AlphaFoldDB at the time of our study. We increased the sensitivity by considering transitive searches (6 iterations), and using newly detected CoBaHMA domains as probes for additional searches. A total of 38444 distinct sequences identifiers were recorded by these searches. Among these identifiers, 28918 had AF2 models. The remaining ones mostly corresponded to UniParc sequences. CoBaHMA-specific features were considered using the AF2 models, restricting the set to a total of 2358 domains corresponding to 2280 sequences, the manual inspection of which then identified 68 false positive domains. Within this whole set, we identified a total number of 2305 (2290 domains from UniClust30 + 15 probes) CoBaHMA domains within 2227 different proteins (2212 sequences from UniClust30 + 15 probes).

Most of the sequences were identified during the two first iterations (see **Supplementary Data 2** for details). Only 8 phyla are represented by more than 20 sequences within the UniClust30 database. All are affiliated to Bacteria. Among them, the Proteobacteria, Cyanobacteria and Bacillota phyla are represented by 1025 (861, 153), 445 (349,78) and 365 (274,89) sequences, respectively. Forty-three, 87 and 40 sequences affiliated to the Actomycetota phylum were identified during the three first iterations. During the whole iterative similarity search process, only one sequence from Eukaryotes was detected during the first transitive search (Chordata, Chondrychtyes class, UniRef100_A0A401TJW7).

**Figure 3** illustrates the conserved sequence patterns of the CoBaHMA domains, derived from the alignment of 1432 sequences representative of the whole domain diversity (see
Material and Methods). The β sheet displays several conserved features, spread over all β strands, except strand β4, which is not always present. By contrast, the two α-helices are highly variable and could not be aligned. First, strand β1 possesses two arginines that are highly conserved: 1266, i.e.88% of the sequences had the two arginines and 1353 (94% of the sequences) had at least one of them. These two arginines are accompanied by another basic residue (arginine or lysine) on the C-terminus of strand β1 as well as a fairly conserved histidine (925 (65%) sequences) in strand β0, and together, form a basic patch at the surface of the β sheet. The full motif HxxxxRxRxR that was originally identified in calcyanins¹² was present in only 27% of the CoBaHMA sequences. A continuum can thus be highlighted in the CoBaHMA family, from sequences that do not have the basic patch to sequences that have the full HxxxRxRxR motif, hinting at the possible existence of sub-families with specific features. Besides this basic patch, strand β1 has a conserved PG motif on its N-terminus. Strand β3 has an array of conserved small amino acids (G/A/T/S) on its N-terminus, as well as an aromatic (Y/F/H) position on its C-terminus, which is oriented toward the hydrophobic core of the CoBaHMA in the 3D structure model. Considering their nature (small or apolar), and/or their position (loops or orientations towards the hydrophobic core), all these conserved amino acids are likely of structural importance. Finally, two polar positions occupied by an asparagine (C-terminus of strand β2) and an acidic residue (C-terminus of strand β3) are also worth mentioning. Interestingly, strand β2, which is the farthest from the basic patch located on strands β0 and β1 appears to have less sequence conservation, except a central position sometimes occupied by a basic residue. This further strengthens the hypothesis that the functional feature of the CoBaHMA is yielded by strands β0 and β1.

## 3.2. Analysis of the full-length sequence communities

Communities of full-length sequences have been defined from a similarity network and the robustness of the affiliation of full length sequences to a given community was achieved by using a refinement method described in the Material and methods (section 2.6).

Communities can be divided into several categories, depending on whether the CoBaHMA domain alone constitutes the entire protein (what we call a "single-CoBaHMA domain protein"), or the protein containing it is longer. Moreover, several scenarios can be distinguished in the latter case, depending on whether or not the other regions of the proteins, apart from the CoBaHMA domain, are already annotated by reference to profiles
contained in domain databases (InterPro (IPR), Pfam) or related to the calcyanin GlyZip motifs (pCALF, see Material and Methods). An additional processing was added, by merging communities sharing identical annotations and/or distinct annotations but corresponding to the same major functional families. We describe the communities using this analysis workflow, adding information about their 3D structures from AlphaFold2 models, as well as conserved motifs identified from multiple sequence alignments (MSA, see **Supplementary Data 3**).

We focused our analysis on the 48 most populated communities, containing at least 5 sequences and amounting to a total of 1410 sequences. Sparsely populated communities are only analyzed on the basis of annotations also found within these large communities.

Most of these 48 large communities are composed of sequences affiliated with different phyla. However, 15 of them (233 sequences) are specific to one bacterial phylum (Figure 4): 6, 5 and 4 communities comprised sequences affiliated to Bacillota (C675, 23 sequences; C521, 20 sequences; C4, 18 sequences; C413, 11 sequences; C397, 8 sequences; C369, 7 sequences), Proteobacteria (C201, 53 sequences; C320, 13 sequences; C562, 10 sequences; C584, 8 sequences; C344, 5 sequences), or Cyanobacteria (C283, 31 sequences; C192, 14 sequences; C20, 7 sequences; C654, 5 sequences) only, respectively.

While most of the large communities do not have any significant functional annotation (non-annotated communities), 20 different domain families from InterPro (IPR) are detected within the full-length sequences of 10 large communities (Supplementary Table 1-A). These families are related to P-type ATPases (10 IPR families), ABC exporters (6 IPR families), type 2 phosphatidylglycerol phosphatases (2 IPR domain families) or HMA with no overlap with the CoBaHMA domains (2 IPR).

Overall, the 48 sequence communities correspond to proteins with different modular organization (Figure 5 and Supplementary Data 4 for the details in each community). These communities may be grouped into categories as follows: small sequences (~100 aa length) containing one CoBaHMA domain only, larger sequences combining a CoBaHMA domain with functionally annotated regions (from IPR or other sources), non-annotated regions, or additional CoBaHMA domains (either 2 or 3). The position of the communities relative to the functional families/phyla and within the network are described in Supplementary Data 5 and 6, respectively.
### 3.2.1. Single-CoBaHMA domain proteins

Eight of the 48 communities (C437, C201, C583, C675, C399, C397, C71, C685) fall into this category, scattered over several phyla (Figure 6). They include a total of 402 sequences, and represent more than 85% of the sequences in communities C437 (181 sequences), C685 (63 sequences), C201 (53 sequences), C583 (48 sequences). Superimposition of the AF2 3D models of their representative members (in which all the core α helices and β strands are predicted with very high/high pLDDT values) indicate that these single-CoBaHMA domain proteins possess extra-regular secondary structures (mostly α helices) at the N- and/or C-terminus of the domain, which pack against the core (Figure 6). Some variations are observed in the β-sheet conserved motifs, with the particular case of community C397 (Figure 6-F), in which the conserved basic amino acids are nor present and the extra-N-terminal helix takes the place of helix α2.

### 3.2.2. CoBaHMA in multidomain proteins

#### • IPR-annotated communities

Only a few large communities show IPR annotations. They exclusively correspond to membrane proteins. We grouped together the IPR categories relating to the same protein families (Supplementary Table 1-A), and also estimated what proportion of the less populated communities ($n<5$, number of communities and number of sequences) can be affiliated to these large protein families. Apart from these major functional families, a limited number of IPR annotations are found in small communities, essentially in singletons (Supplementary Table 1-B)

1) **P-type ATPases.** P-type ATPases represent the bulk of the set, amounting to 216 sequences, out of a total of 516 sequences over the whole set of communities (Supplementary Table 1). P-type ATPases are composed of a common core of three conserved domains: (i) a discontinuous transport-domain (T-domain) made of six membrane-spanning helices (M1 to M6) providing the substrate translocation pathway, (ii) an ATP-binding domain (ATPBD - between M4 and M5), which includes the nucleotide-binding domain (N-domain) and the phosphorylation domain (P-domain), and (iii) an actuator domain (A-domain, between M2 and M3), which is believed to transmit changes in the ATPBD to the T-domain and to drive
dephosphorylation<sup>11</sup>. Two additional domains can complete this common core, depending on the considered P-type ATPase subset: (i) the **S-domain**, which is an auxiliary membrane unit providing support to the T domain and is located at various positions in the sequence (N- or C-terminal relative to the T domain); (ii) **regulatory (R) domains**, which are located at the N-terminus and/or C-terminus and act as intramolecular inhibitors, sensors for transported cations and/or regulators for cation affinities<sup>11</sup>. Transport is accomplished via a so-called Post-Albers cycle in which phosphorylation of a conserved aspartate residue in the ATPBD causes the protein to cycle between high (E1)- and low (E2)-affinity ion-binding states. InterPro entries (IPR, **Supplementary Table 1-A**) are available to annotate the P-type ATPases over their full-length common core (IPR001757) or domains (IPR023298: T-domain; IPR008250: A-domain; IPR023299: N-domain; IPR044492, IP036412, IPR023214: HAD/HAD-like), while other IPRs provide annotations for specific P-type subsets (e.g. IPR027256 for P<sub>1B</sub>-type, IPR004014 and IPR006068 for cation-transporting P-type ATPases N-terminal and C-terminal, respectively). IPR entries specific of HMA domains (IPR036163 and IPR006121) are also found, outside the limits of the CoBaHMA domains, as accompanying some P<sub>1B</sub>-type ATPases.

Seven communities with at least 5 sequences and scattered over several phyla (**Figure 9**) are annotated as P-type ATPases (**Supplementary Table 1-A**). Five out of the seven communities (**C366, C429, C525, C140, C20**) belong to the **P<sub>1B</sub>-type**. Indeed, the AlphaFold2 models of their representative sequences (**Figure 7**) include a S-domain specific to this subset, comprising two transmembrane helices, a long and curved MA helix and a kinked MB helix with an amphipathic MB' segment at the cytoplasmic membrane interface, lining the ion entry point<sup>53,54</sup>. Four out of these 5 communities (**C366, C429, C525, C140**) match the IPR027256 (P<sub>1B</sub>-type) profile. The five communities differ by the architecture of their whole proteins: the biggest community, **C366** (115 sequences, representative member: A0A3P1Y6T0, **Figure 7-A**), as well as **C429** (7 sequences, representative member: A0A7I7MP94, not represented in **Figure 7**) have a N-terminal CoBaHMA domain. Community **C525** (36 sequences, representative member: A0A5C7KBI4 - **Figure 7-B**) is characterized by a N-terminal CoBaHMA + HMA couple. Community C140 (18 sequences, representative member: A0A1Y6CVZ1, **Figure 7-C**) contains a tandem of CoBaHMA domains forming a continuous beta-sheet, with two additional strands in the sequence linking them. However, only the C-terminal CoBaHMA domain was detected by our search, while the N-terminal one lacked most of the basic residues. Last, the **C20** community (7 sequences, representative member: A0A3S1CNK6, **Figure 7-D**), specific to
cyanobacteria, lacks the P-domain and has degenerated consensus motifs in domains A and N, in contrast to the other communities which preserve these critical sequences (domains A ([TS]-G-[DE]), P (DKTGT) and N (HP)) **(Supplementary Data 3)**.

In addition, among the 7 communities annotated as P-type ATPases, the **C413** community (11 sequences, representative member: R1CS5, **Figure 7-E**) possesses a single N-terminal CoBaHMA domain but has an atypical MA-MB segment, which does not match the usual topology encountered in P<sub>1B</sub>-type ATPases and could not be modelled accurately.

Finally, the community **C556** (22 sequences, representative member: A0A7C4R520, **Figure 7-F**) has also a CoBaHMA domain in the N-terminus but do not belong to the P<sub>1B</sub>-type. Instead, it is annotated by cation N-terminal (IPR004014) and cation C-terminal (IPR006068) profiles (**Supplementary Table 1-A**), which are found in several cation-transporting ATPases (Na<sup>+</sup>, K<sup>+</sup>, Ca<sup>2+</sup>). Inspection of the AlphaFold2 model indicated a conserved calcium binding site in the T-domain<sup>55,56</sup>.

Members of the P<sub>1B</sub>-type subsets were described heretofore as specific to the translocation of heavy metal ions. They are divided into several groups based on conserved sequence motifs (in the unwound part of M4, but also in M5 and M6) and the selectivity of the transported metal ion<sup>57</sup>. The six communities (**C366**, **C429**, **C525**, **C140**, **C20**, **C413**) of P<sub>1B</sub>-type ATPases highlighted here possess conserved motifs in the unwound part of M4, which differ from one community to the other. The biggest community **C366** can be divided into two sub-communities, according to these motifs (**Figure 7-A**): (i) Part of the sequences, such as *D8F5K4*, has a conserved C-P-C motif (dotted box in **Figure 7-A**), typical of heavy metal binding sites; (ii) other sequences, such as the representative sequence A0A3P1Y6T0, contain a characteristic conserved motif including an aspartate as well as a basic residue (D-[YF]-x-[TC]-x(2)-[KRH]). Both sub-communities have the same basic signature in their CoBaHMA domain, including a histidine in stand β0. Community **C429** has a S-[SC]-P-C motif, similar to the **C366** *D8F5K4* to which it is linked. A C-P-C motif is also found conserved in community **C525**, while community C170 possesses a conserved M4 motif also including acidic and basic residues (N-X-D-X-G-T-G-I-R). Last, the **C413** community has no strictly conserved residue in the unwound part of M4, except from a central proline.

In conclusion, our results indicate that CoBaHMA domains form a novel family found frequently in association with P<sub>1B</sub>-type ATPases, similarly to HMA domains. However,
432

sequence signatures of heavy metal binding in M4 are not systematically found in these P<sub>1B</sub>-type ATPases, giving way to other signatures, including conserved acidic and basic residues and suggesting that P<sub>1B</sub>-type ATPases are not exclusively transporting heavy metals.

433

434

435

436

**2) ABC exporters.** CoBaHMA domains are also found in the N-terminus of type I ABC exporters (17 out of 31 sequences over the whole set of communities). They are present in two large communities: **C538** (*representative member UniProt A0A2W6BRH6*, 11 sequences) and **C520** (*representative member UniProt E3FPA8*, 6 sequences).

437

438

439

440

Type I ABC exporters, formerly known as type IV exporters<sup>58</sup>, transport a wide variety of substrates across membranes. They consist in a TMD with six transmembrane (TM) helices and a Nucleotide Binding Domain (NBD), which form homo- or hetero-dimers, with a swapped arrangement of two TMs. Three InterPro IPR are associated with these two communities: IPR036640, an ABC transporter type I of the transmembrane domain superfamily; IPR027417, a P-loop containing nucleoside triphosphate hydrolase, and IPR039421, a type 1 protein exporter, encompassing both the transmembrane domain (TMD) and the nucleotide binding domain (NBD). The experimental 3D structures closest to the AF2 models of the C538 and C520 representative sequences (FoldSeek searches) are those of bacterial ABC exporters involved in the transport of various substrates, including lipid A (MsbA, pdb 7PH4), peptides (TmrAB, pdb 6RAI) or multiple drugs (Sav1866, pdb 2HYD). The CoBaHMA-containing ABC exporter sequences exhibit canonical ABC conserved motifs in the NBD (Walker A, Walker B, ABC signature), suggesting that they are active transporters (**Supplementary Data 3**). In contrast to other communities with CoBaHMA domains, the ABC exporter communities do not have the conserved histidine in strand β0, while strands β0 and β1 include several basic amino acids (**Figure 8-A**). Linkers of variable length separate the N-terminal CoBaHMA domain from the TMD. These are predicted with lower pLDDT values as random coil or most often, as a TMD hairpin (e.g. A0A2W6BRH6, community C538) or both (e.g. A0A6G4WXH4, community C455 or A0A7V8NHJ8, community C499), depending on the ABC exporter sequence. It is precisely the length of the linker that makes the difference between the two most populated communities, **C538** and **C520**, **Figure 8-A**).

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

Worth noting, a group of small communities, united under the common denominator EcsC proteins (IPR024787: **C663**: 4 sequences, *representative member: A0A552EVV8*; **C684**: 1

463
sequence, A0A098TP70; C83: 1 sequence, A0A6H2NMM5, C52: 1 sequence, Q606U9) is
related to ABC transport systems. Indeed, in *Bacillus subtilis*, EcsC is found in an operon with
EcsA and EcsB, which are components of an ABC transport system⁵⁹. The AF2 model of the
EcsC domain folds as an *a priori* soluble bundle of TM helices, with most of the amino acids
characterized by low pLDDT values (Figure 8-B). A FoldSeek search did not highlight any
significant similarity with known 3D structures, suggesting that this domain adopts an as yet
uncharacterized fold. The EcsC CoBaHMA domains possess the characteristic His/Arg
signature in the first two β strands.

472

C) **Type 2 phosphatidylglycerol phosphatases (PAP2).** CoBaHMA domains are present in the N-terminus of some type 2 phosphatidylglycerol phosphatases (PAP2) in Cyanobacteria. Community C419 (representative member UniProt A0A3S1IWR7) contains 22 sequences, whereas single members are found in 8 additional communities (C16, C311, C418, C542, C617, C629, C642, C643), i.e., amounting in total to 30 sequences. PAP2 sequences are described by two InterPro entries: IPR036938 and IPR000326, both entries being defined as phosphatidic acid phosphatase type 2/haloperoxidase. Integral membrane proteins from the PAP2 family dephosphorylate a variety of compounds, including lipids and carbohydrates ⁶⁰. They consist in a core TMD, with six tightly packed TM helices connected by extramembrane loops, two of which interacting together to form the catalytic site (Figure 8-C). The sequences of the CoBaHMA-containing PAP2 belong to the lipid phosphatase/phosphotransferase (LPT) family, as they all contain the conserved tripartite active site motif (KX₆RP---PSGH---SRX₅HX₃D) ⁶¹ (Supplementary Data 3). Members of this family modify several types of lipids in Gram-negative bacteria, e.g. phosphatidylglycero-phosphate (PGP) for PgpB ⁶², or lipid A for LpxE ⁶³. The AF2 model of the representative sequence resembles the 3D structure of *B. subtilis* bsPgpB (pdb 6FMX(A)-5JKI(A), El Ghachi et al. 2017; FoldSeek TM-score 0.87, 26.7% identity), which contains eight TM α-helices, six of them (α1,α4-α8) being tightly packed, while the α2 helix is amphiphilic, lying at the surface of the lipid bilayer on the active site side (Figure 8-C). The linker separating the CoBaHMA domain, located in the intracellular milieu, from the terminal TM α1-helix is variable. It is predicted as a random coil or even as integrating additional TM α-helices (e.g. A0A433NH73, community C542), always with very low pLDDT values. Again, the PAP2 CoBaHMA domains possess the characteristic His/Arg signature in the first two β strands.
### D) Diacylglycerol kinases

Although not included in large communities, two small communities of cyanobacterial proteins, **C515** (1 sequence, A0A0S3UCN3) and **C553** (2 sequences, representative member: A0A841V906), caught our attention. They match several IPR profiles (IPR045540; IPR017438; IPR016064; IPR005218; IPR001206; IPR004363) related with bacterial **diacylglycerol (DAG) kinases**. These enzymes convert DAG, formed by the turnover of membrane phospholipids, to phosphatidic acid <sup>64</sup>. In these proteins, the CoBaHMA domain is located at an unusual C-terminal position (**Figure 8-D**), while the N-terminal domain corresponds to the catalytic DAK kinase (FoldSeek matches with the putative DAG kinase from *Bacillus anthracis* (pdb 3T5P(B), TM-score 0.83, 25 % identity, Hou et al. unpublished) and the DAG kinase DgkB from *Staphylococcus aureus* (pdb 2QVL(A), TM-score 0.81, 22 % identity <sup>64</sup>). The 3D structure of DgkB has a two-domain architecture, similar to that found in *E. coli* YegS, which phosphorylates *in vitro* phosphatidyl glycerol <sup>65</sup>. Members of the **C515** and **C553** families share the conserved P-loop ($\phi$-x-x-G-G-D-G-T-$\phi$, where $\phi$ represents a hydrophobic amino acid), but exhibit slight difference in the two other conserved motifs, as described in <sup>64</sup> (($\phi$-$\phi$-x-N-P-x-S/A-G instead of $\phi$-$\phi$-x-N-P-x-S-G and $\phi$-$\phi$-P-x-G-T-x-N-A-$\phi$-x-N instead of $\phi$-$\phi$-P-x-G-T-x-N-D-$\phi$-x-R; side and top of the nucleotide-binding site, respectively). All three sequences share a common domain in between the DAG kinase and the CoBaHMA domains, modeled with very low pLDDT values as a long helix (A0A841V906) or a two-helix hairpin (A0A0S3UCN3). The **C515** single member community (A0A0S3UCN3) differs from the two other sequences of the **C553** community by an additional C-terminal domain, which is related to the GlyZip motif described below. The DAGK CoBaHMA domains possess the characteristic His/Arg signature in the first two $\beta$ strands.

### • Other annotated, non-IPR, communities

#### A) HMA-2

Eight hundred and thirty-two CoBaHMA domains overlap the Pfam profile HMA-2 (PF19991). This profile is described as distantly related to HMA domains in its N-terminal part, containing in particular the highly conserved histidine we also highlighted in this study. Not all the CoBaHMA domains match the N-terminal part of the HMA-2 profile, indicating that proteins matching this profile constitute a subset of the CoBaHMA family. However, the HMA-2 profile
528 is larger than CoBaHMA domains, with a total length of 180-190 amino acids, and includes at
529 its C-terminal part a conserved region generally predicted as two contiguous helices. Matches
530 to this C-terminal region are observed for sequences in 11 communities (C75, 81 sequences,
531 representative member: A0A564ZMZ6; C393, 62 sequences, representative member: A0A1Z4FYZ0;
532 C141, 46 sequences, representative member: A0A1J1CTN7; C586, 24 sequences, representative
533 member: C9KNI5; C329, 23 sequences, representative member: A0A7Y6UHT; C521, 20 sequences,
534 representative member: A0A366XQZ5; C30, 17 sequences, representative member: A0A2U3KUJ9;
535 C562, 10 sequences, representative member: A0A4R3M3I3; C294, 7 sequences, representative
536 member: A0A2V7B4L6, C344, 5 sequences, representative member: A0A4P2PVK5; C5, 5 sequences,
537 representative member: A0A662ZLY5). For communities having a C-terminal region associated
538 with high AF2 pLDDT values (≥70), such as C393, the two helices, often predicted as
539 transmembrane segments, pack together to form a hairpin (Figure 9-A). However, no obvious
540 similarity with any known 3D structure could be detected by FoldSeek for this case, outside
541 helix hairpins belonging to larger assemblies.

542 Matches with the HMA-2 profile were also detected in other communities, however limited
543 to the N-terminal CoBaHMA domain. We analyzed the regions C-terminal to the HMA-2 N-
544 ter/CoBaHMA domain, especially for detecting possible distant relationships to the HMA2-
545 Cter profile. Three communities C202 (56 sequences, representative member: A0A5C7T941),
546 C320 (13 sequences, representative member: A0A0X8JQ54) and C584 (8 sequences,
547 representative member: A0A3B9QA35) also possess a hairpin of two helices, often predicted
548 as transmembrane segments, with conserved amino acids. However, they differ from those
549 defining the HMA2-Cter profile (illustrated with the representative sequence of the C202
550 community on Figure 9-B). In particular, the C202, C320 and C584 C-terminal regions are
551 characterized by highly conserved histidine residues, together with basic (arginine, lysine)
552 residues. Moreover, a FoldSeek search against PDB detected significant similarities (TM-score
553 0.82) between the C202 representative sequence and the long alpha-hairpin domain of a
554 manganese/iron superoxide dismutase (pdb 4BR6), which forms a four-helix bundle through
555 protein dimerization and provides histidine residues to the ion-binding site at the interface
556 with the preceding domain ⁶⁶. In community C331 (16 sequences, representative member:
557 Q8YVH2), in which proteins possess two CoBaHMA domains, the first HMA-2 N-ter/CoBaHMA
558 is also followed by a helical hairpin, predicted with low pLDDT values. This helical hairpin
559 contains conserved charged and aromatic amino acids. The second CoBaHMA domain is also
followed by a helical region, although less defined (Figure 9-C). The representative sequence (A0A6M0J010) of community C341 (19 sequences) has a C-terminal region with three non-packed alpha-helices, also predicted with very low pLDDT values and containing conserved basic and acidic residues (Figure 9-D), while the representative sequence (A0A522V264) of community C444 (100 sequences) has two non-assembled alpha-helices, with less amino acid conservation. Representative sequences from communities C725 (A0A6F9WUC5, 12 sequences) and C352 (A0A7Y4FMF1, 31 sequences) are apparently disordered (Figure 9-E), but contain conserved charged residues accompanying hydrophobic clusters, suggesting a hidden fold (Supplementary Data 3). Interestingly, a cyanobacterial community (C283, 31 sequences, representative sequence: B1WT30) has the HMA2-C-ter region at its N-terminus, preceding the HMA2-Nter/CoBaHMA domain. This N-terminal HMA2-C-ter region is also folded as a helical hairpin, with conserved charged/polar residues (Figure 9-F).

Finally, CoBaHMA domain-containing sequences belonging to the community C678 (12 sequences, representative member: A0A7W8FDY5), which do not match the HMA2 profile, also contain a helical hairpin with two conserved histidine residues in the second helix (Figure 9-H). These features are similar to the ones observed in the C202, C320 and C584 communities. A helical hairpin, with highly conserved charged amino acids, is also present in three other non-HMA2 communities: C106 (9 sequences, representative member: A0A7C2VAZ9; Figure 9-I), C578 (7 sequences, representative member: A0A1H4BM11; Figure 9-J) and C487 (50 sequences, representative member: H8GQW7; Figure 9-K).

## B) Calcyanins: a (GlyZip)3 motif detected by a dedicated tool (pCALF)

Community C192 (14 sequences) corresponds to calcyanins, in which the CoBaHMA domain was first identified. Some of the CoBaHMA domains of this community match the HMA2 N-ter profile described above. Calcyanins contain a C-terminal domain, consisting in a three-fold repeat of a large glycine-zipper (GlyZip) motif. This large GlyZip motif itself corresponds to a duplicated smaller glycine zipper motif, interrupted in its middle part by a conserved Gly-Pro dipeptide (Supplementary Data 7). AF2 modeled this GlyZip motif as a hairpin of tightly packed helices, consistent with the presence of conserved glycine residues repeated every four residues (Figure 9-G)¹⁴,⁶⁷. The low/very low pLDDT values may be due to the very large sequence distance between this GlyZip motif and known hairpins of this type, present in
592

different architectures (as explored with FoldSeek). However, AF2 fails to assemble these
GlyZip motifs in a consistent way.

593

594

595

• Non-annotated communities

596

Last, very few communities with n≥5 other than those described above have been detected.
597
Protein segments associated with CoBaHMA domains correspond to disordered or ordered
598
regions. The order (foldability) in these regions was assessed by examining AF2 predictions, in
599
particular the segments associated with high/very high pLDDT values (≥0.7), as done for the
600
HMA-2 N-ter containing proteins. By this way, we also retrieved helical hairpins in other than
601
HMA-2 CoBaHMA communities (Figure 9-H to -K). In contrast, low values of pLDDT are
602
sometimes indicative of disorder, although in some cases, low pLDDT values are associated
603
with genuinely well folded regions (predicted as folded or in random coil), but for which the
604
prediction cannot be supported. For example, this is the case of new folds and sequences
605
lacking homologs 37,38. One way to evidence these" hidden" folded domains is to assess
606
foldability using Hydrophobic Cluster Analysis (HCA) 37,38. Therefore, we investigated
607
unannotated sequences by combining AF2 structure predictions and, when needed, HCA
608
analyses of the protein sequences.
609

597

598

599

600

601

602

603

604

605

606

607

608

609

610

The most populated community (C233; 39 sequences; representative sequence: U2SKL7) is
611
predicted by AlphaFold2 as a CoBaHMA domain with a C-terminal extension comprising three
612
strands completing the core beta-sheet (Figure 10-A). A four-helix bundle predicted with high
613
pLDDT values is inserted in between these the CoBaHMA domain and this C-terminal
614
extension. Interestingly, a FoldSeek search of this four-helix bundle, which contains strictly
615
conserved histidine, arginine and aromatic residues, indicated a possible structural
616
relationship with the MA-MB-M1-M2 block of P1B-type ATPases (pdb 4UMWT-score 0.54,
617
11.3 identity, Figure 10-A). The MB kink (resulting in MB') is not present in the C233 four-helix
618
bundle, whereas the basic residues (histidine and arginine) are located at the entry site of the
619
P1B-type ATPase.
620

611

612

613

614

615

616

617

618

619

620

The C4 community (18 sequences, representative sequence: A0A1H0C0W7) is also predicted
621
by AlphaFold2 as composed of a N-terminal CoBaHMA domain and a four-helix bundle (Figure
622
10-B), also with conserved amino acids (especially arginine and histidine residues) located at
623
the tip of the bundle. However, in this case, no significant structural similarity was found

621

622

623
624

between the representative member of the community and any experimental 3D structures
using FoldSeek.

625

626

A third community (C93, 6 sequences, representative sequence: A0A552LCK1) possesses a C-terminal CoBaHMA domain, preceded by a globular domain, with conserved residues, but which does not share obvious similarity with any available experimental 3D structure (FoldSeek search) (Figure 10-C).

627

628

629

630

Finally, we also examined the C654 community (5 sequences, representative sequence: A0A0M0SS18). It is characterized by a C-terminal HMA-2 N-ter/CoBaHMA domain, preceded by a tandem of CoBaHMA-like domains (devoid of the conserved basic signature). However, it did not contain any other folded domain based on AlphaFold2 modelling and HCA analysis (Figure 10-D).

631

632

633

634

635

636
## 4. Discussion

The large-scale predictions of 3D structures now enabled by AI-based approaches allow to functionally annotate large sets of proteomes at the amino acid level, and identify new folds (e.g. ⁶⁸). The 3D models provided by AlphaFold2 via a dedicated database ²², connected to UniProt ²⁹, offer an unprecedented tool for extending the exploration of the universe of protein domains and studying their evolutionary trajectory. Here, we have taken advantage of this large-scale structural information, combined with sequence similarity search and clustering tools, to identify a new family of domains called CoBaHMA. This domain family shares a common evolutionary origin with HMA domains, as evidenced by the signature of a common hydrophobic core. However, CoBaHMA can be discriminated from HMA based on an additional, external β strand (called β0) and, in many cases, a specific sequence signature, conferring a positive electrostatic charge on one side of the beta sheet, which is likely to be associated with a specific function. The AlphaFold2 models provided a structural constraint throughout the workflow we built, enabling a fine discrimination between CoBaHMA domains and the rest of the very large HMA superfamily. Besides providing information about a novel family of domains, our study illustrates how evolution may operate within a superfold to provide broad functional diversity.

This study may have some limitations. Indeed, the structural features we have automatically defined lightly suffer from the definition of cutoffs, which does not rule out the possibility that highly divergent members of the CoBaHMA with large loops between the two first β strands may be overlooked. The methodology developed is also dependent on the availability and accuracy of AF2 models. Therefore, the CoBaHMA family described in this work constitutes a minimum set as we have considered a non-redundant bank of sequences (UniRef30). However, even with this possible extension, CoBaHMA domains are only present in a restricted set of proteins, suggesting a specific function within different protein families limited to certain species or phyla.

Further prediction of the CoBaHMA-specific function, or at least the biological environment in which it is performed, can be aided by analyzing the domain architecture of the proteins containing the CoBaHMA domain. Here, deciphering this architecture was again aided by the AlphaFold2 predictions, combined with domain database (InterPro) annotations. A large part of the CoBaHMA communities corresponds to single CoBaHMA domain proteins. This is
669 reminiscent of the single-HMA domain proteins, behaving as chaperones⁶⁹. CoBaHMA
670 domains are also found in association with a limited number of protein families, which mostly
671 correspond to membrane proteins, at least for those annotated through InterPro profiles and
672 predictors of transmembrane segments. Moreover, CoBaHMA domains appear to be specific
673 to bacteria, in contrast to HMA domains which are found in bacteria, archaea and eukaryote.
674 Overall, These observations suggest a membrane-related function specific to bacteria for
675 CoBaHMA..

676

677 Like HMA domains, CoBaHMA domains are particularly abundant in P₁B-type ATPases. The
678 latter are commonly defined as integral membrane proteins that couple ATP hydrolysis to the
679 transport of metal cations, such as copper, zinc and cobalt ⁵⁷. The specificity of P₁B-type
680 ATPases towards heavy metals is linked to conserved motifs in the middle of the fourth
681 transmembrane helix (TM4). These motifs directly coordinate the ion through
682 cysteine/histidine side chains. Besides, soluble N- and C-terminal metal-binding extensions
683 (known under the generic term Heavy Metal Binding Domains (HMBDs)), also rich in
684 cysteine/histidine and including HMA domains, seemingly play a regulatory role ⁵³,⁵⁴,⁷⁰. In
685 particular, HMBDs interact with the amphipathic helix MB', lying at the membrane-cytosol
686 interface at the end of a P₁B-specific MA and MB membrane hairpin ⁷¹. This amphipathic MB'
687 helix is connected to the high affinity ion-binding site through a conserved electronegative
688 funnel. HMBDs also interact with the cytosolic domains (A and P domains), thus playing a
689 potential regulatory role by interfering with conformational changes coupling ATP hydrolysis
690 with ion transport across the membrane ⁷¹. Here, we show that P₁B-type ATPases are not
691 restricted to heavy metals. Indeed, the proteins identified here share a P₁B-specific MA and
692 MB membrane hairpin but contain CoBaHMA domains instead of the usually encountered
693 HMA domains and have conserved motifs in TM4 different from those coordinating heavy
694 metals. These motifs vary depending on the considered community, often including charged
695 (acidic and/or basic) or polar (serine/threonine) residues. Interestingly, one community
696 contains the TM4 CPC motif, typical of heavy metal transport, together with a CoBaHMA
697 domain only, and no HMA domain at its N-terminus. This suggests that the coupling of HMA
698 with TM4 Cys-rich motif is not necessary as initially thought. Moreover, a large
699 sequence/structure diversity is observed at the level of the MA/MB hairpin, probably linked
700 to the substrate diversity of CoBaHMA-containing P₁B-type ATPases, as also evidenced by the
diversity of the M4 conserved motifs. Finally, it is worth noting that CoBaHMA domains are
also found associated with members of the P₂A-type ATPases (SERCA, Ca²⁺ ATPases), and are
thus not restricted to the P₁B-type subgroup.

The question remains as to what are the substrate specificities of these P-type proteins
associated with CoBaHMA domains, and what is the role of the CoBaHMA domains in this
specific modular organization. A regulatory role similar to that played by HMA could be
expected, supported by the fact that some AF2 models displayed significant interfaces with A
domains, involving amino acids outside the conserved basic patch (Supplementary Data 8).
The identity of the ligands for these charged amino acids remains to be explored.

710

Accessory domains provide an additional, often regulatory effect on the functions of ATP-
binding cassette (ABC) exporter cores, which are formed by transmembrane domains (TMDs)
and nucleotide-binding domains (NBDs) ⁷². For instance, the cytosolic Cystathionin Beta
Synthase (CBS) domains, at the C-terminus of the osmoregulatory OpuA, inhibits the
transporter activity by binding to cyclic-di-AMP ⁷³. This protein is gated by ionic strength,
which modulates the interaction of positively charged amino acids in the NBDs with negatively
charged lipids ⁷³. Although, like for the P-type ATPases, the specific function of CoBaHMA
domains in this ABC context remains to be discovered, a number of points can be considered.

First, the ABC transporters with the highest sequence identities (~30 %) include lipid
transporters, including MsbA, suggesting that (i) the CoBaHMA-domain-containing ABC
transporters might be involved in lipid transport, and (ii) the CoBaHMA domain might be
directly involved in the uptake of lipids. Second, the specific position of the domain, N-terminal
to the NBD, places it at the right location to interact with the polar heads of lipids, as observed
for instance with the lasso domain found in some ABCC transporters such as the Cystic Fibrosis
Transmembrane conductance Regulator (CFTR) protein (ABCC7) (⁷⁴ for a review). This suggests
a specific role in contacting the membrane via the conserved basic patch at the surface of the
domain. The predicted presence of additional TM helices between the CoBaHMA domain and
the TMD in most of the ABC communities, like in the ABCC transporters ABCC1 (MRP1) and
ABCC8 (SUR1) ⁵⁸, might serve an additional regulatory purpose. This "lipid hypothesis" is also
interesting to consider with regard to the function of CoBaHMA domain in the context of P₁B-
type ATPases, particularly as specific transport of lipids is carried out by another class of P-
type ATPases (P₄-type, ⁷⁵,⁷⁶).
733

734

The hypothesis of the CoBaHMA domain serving as a binding module for positively charged lipids (phospholipids) in bacteria is appealing considering the wide knowledge about phospholipids-binding domains in eukaryotes. Indeed, the tray of basic amino acids offered by CoBaHMA domains resembles that observed in C2 domains for instance, which interact with membranes in a Ca²⁺-dependent manner through a polybasic cluster, with specificity to phosphatidylinositol-4,5-bisphosphate ⁷⁷,⁷⁸.

735

736

737

738

739

740

The phosphatidylinositol phosphates that are largely recognized in eukaryotic membranes are also the targets of some bacterial proteins acting as effectors or toxins (e.g. the lipid raft targeting domain of the *Bordetella* pathogens ⁷⁹, also see ⁸⁰ for a review). However, bacterial membranes are distinct in lipid composition from eukaryotic membranes, and their lipid-binding modules are far less well known. Phosphatidylglycerol (PG) might be a target for the CoBaHMA domain. This phospholipid is present in both Gram- and Gram+ bacteria, and plays a central role in the synthesis of cardiolipin (CL, diphosphatidylglycerol), lysophosphatidylglycerol (LPG) and oligosaccharides ⁸¹. Phosphatidic acid (PA) is another potential candidate. It is linked to the activity of diacylglycerol (DAG) kinase, and serves as a precursor for glycerolipids ⁸². An appealing hypothesis is that, in addition to being specifically recognized by the CoBaHMA domains, these phospholipids could be transported by membrane systems in which CoBaHMA is included (e.g. ABC exporter), a mechanism which could contribute to the general lipid homeostasis. However, such hypotheses remain highly speculative and need to be extensively tested.

741

742

743

744

745

746

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763
One of the outstanding features of our domain grammar analysis was the co-occurrence of the CoBaHMA domain not only with already annotated, membrane-specific domains, but also with hairpins of two consecutive helices. These helical hairpins show both a conserved structural motif as revealed by AF2 models, and a wide variety of sequences: some include a lot of strong hydrophobic amino acids, as in the HMA-2 C-ter profile, and are predicted as forming transmembrane segments; others include small but also globally apolar residues. However, most of them include conserved, charged residues, in particular histidine and basic residues. This suggests that these hairpins may serve as platforms on which amino acids can be grafted to interact with specific ions or ligands. From an evolutionary point of view, it is tempting to speculate that these hairpins can be used as basic units for integrating more complex architectures, such as ABC exporter TMDs or those present in calcyanins (three repeats of a glycine-zipper helical hairpin). These calcyanin glycine-zippers are structures characterized by very compact assemblies due to the presence of glycine every 4 residues, but it remains yet to be specified whether they are soluble or membrane-bound. One open question is to what extent the MA-MB hairpin specific to P<sub>1B</sub>-type ATPases may have evolved from this basic module. Finally, from a methodological point of view, it would be interesting to consider this CoBaHMA-specific grammar to improve sequence similarity searches, as done for instance by Terrapon et al.<sup>87</sup> and Faure and Callebaut<sup>18</sup> or, more recently, by Buchan and Jones<sup>88</sup> using natural language word embedding techniques.
785 1. Orengo CA, Jones DT, Thornton JM. Protein superfamilies and domain superfolds. *Nature*. 1994;372(6507):631-634.

787 2. Chitturi B, Shi S, Kinch LN, Grishin NV. Compact Structure Patterns in Proteins. *J Mol Biol*. 2016;428(21):4392-4412.

789 3. Kolodny R. Searching protein space for ancient sub-domain segments. *Curr Opin Struct Biol*. 2021;68:105-112.

791 4. Chandonia JM, Guan L, Lin S, Yu C, Fox NK, Brenner SE. SCOPe: improvements to the structural classification of proteins - extended database to facilitate variant interpretation and machine learning. *Nucleic Acids Res.* 2022;50(D1):D553-d559.

794 5. Caetano-Anollés G, Caetano-Anollés D. An evolutionarily structured universe of protein architecture. *Genome Res.* 2003;13(7):1563-1571.

796 6. Thornton JM, Orengo CA, Todd AE, Pearl FM. Protein folds, functions and evolution. *J Mol Biol.* 1999;293(2):333-342.

798 7. Grishin NV. Fold change in evolution of protein structures. *J Struct Biol*. 2001;134(2-3):167-185.

800 8. Jung J, Lee B. Circularly permuted proteins in the protein structure database. *Protein Sci.* 2001;10(9):1881-1886.

802 9. Arnesano F, Banci L, Bertini I, et al. Metallochaperones and metal-transporting ATPases: a comparative analysis of sequences and structures. *Genome Res.* 2002;12(2):255-271.

805 10. Bull PC, Cox DW. Wilson disease and Menkes disease: new handles on heavy-metal transport. *Trends Genet.* 1994;10(7):246-252.

807 11. Palmgren MG, Nissen P. P-type ATPases. *Annu Rev Biophys*. 2011;40:243-266.

808 12. Benzerara K, Duprat E, Bitard-Feildel T, et al. A New Gene Family Diagnostic for Intracellular Biomineralization of Amorphous Ca Carbonates by Cyanobacteria. *Genome Biol Evol.* 2022;14(3).

811 13. Kim S, Chamberlain AK, Bowie JU. Membrane channel structure of Helicobacter pylori vacuolating toxin: role of multiple GXXXG motifs in cylindrical channels. *Proc Natl Acad Sci USA.* 2004;101(16):5988-5991.

814 14. Kim S, Jeon TJ, Oberai A, Yang D, Schmidt JJ, Bowie JU. Transmembrane glycine zippers: physiological and pathological roles in membrane proteins. *Proc Natl Acad Sci USA.* 2005;102(40):14278-14283.

817 15. De la Concepcion JC, Franceschetti M, Maqbool A, et al. Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen. *Nat Plants.* 2018;4(8):576-585.

820 16. Jiang D, Zhao Y, Fan J, et al. Atomic resolution structure of the E. coli YajR transporter YAM domain. *Biochem Biophys Res Commun.* 2014;450(2):929-935.

822 17. Jiang D, Zhao Y, Wang X, et al. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. *Proc Natl Acad Sci U S A.* 2013;110(36):14664-14669.

825 18. Faure G, Callebaut I. Identification of hidden relationships from the coupling of hydrophobic cluster analysis and domain architecture information. *Bioinformatics.* 2013;29(14):1726-1733.

828 19. Jin J, Xie X, Chen C, et al. Eukaryotic protein domains as functional units of cellular evolution. *Sci Signal.* 2009;2(98):ra76.

830 20. Vogel C, Bashton M, Kerrison ND, Chothia C, Teichmann SA. Structure, function and evolution of multidomain proteins. *Curr Opin Struct Biol.* 2004;14(2):208-216.
832 21. Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. *Nature*. 2021;596(7873):583-589.

834 22. Varadi M, Anyango S, Deshpande M, et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. *Nucleic Acids Res.* 2022;50(D1):D439-d444.

837 23. Mirdita M, von den Driesch L, Galiez C, Martin MJ, Söding J, Steinegger M. Uniclust databases of clustered and deeply annotated protein sequences and alignments. *Nucleic Acids Res.* 2017;45(D1):D170-d176.

840 24. Steinegger M, Meier M, Mirdita M, Vöhringer H, Haunsberger SJ, Söding J. HH-suite3 for fast remote homology detection and deep protein annotation. *BMC Bioinformatics*. 2019;20(1):473.

843 25. Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. *Biopolymers*. 1983;22(12):2577-2637.

845 26. Touw WG, Baakman C, Black J, et al. A series of PDB-related databanks for everyday needs. *Nucleic Acids Res.* 2015;43 part (Database issue):D364-368.

847 27. Rost B, Eyrich VA. EVA: large-scale analysis of secondary structure prediction. *Proteins*. 2001;Suppl 5:192-199.

849 28. Ettema TJ, Huynen MA, de Vos WM, van der Oost J. TRASH: a novel metal-binding domain predicted to be involved in heavy-metal sensing, trafficking and resistance. *Trends Biochem Sci.* 2003;28(4):170-173.

852 29. UniProt: the Universal Protein Knowledgebase in 2023. *Nucleic Acids Res.* 2023;51(D1):D523-d531.

854 30. Shen W, Ren H. TaxonKit: A practical and efficient NCBI taxonomy toolkit. *J Genet Genomics*. 2021;48(9):844-850.

856 31. Jones P, Binns D, Chang HY, et al. InterProScan 5: genome-scale protein function classification. *Bioinformatics*. 2014;30(9):1236-1240.

858 32. Blum M, Chang HY, Chuguransky S, et al. The InterPro protein families and domains database: 20 years on. *Nucleic Acids Res.* 2021;49(D1):D344-d354.

860 33. Manriquez-Sandoval E, Fried SD. DomainMapper: Accurate domain structure annotation including those with non-contiguous topologies. *Protein Sci.* 2022;31(11):e4465.

863 34. Hallgren J, Tsirigos KD, Pedersen MD, et al. DeepTMHMM predicts alpha and beta transmembrane proteins using deep neural networks. *bioRxiv.* 2022;2022.004.2008.487609.

866 35. Bitard-Feildel T, Lamiable A, Mornon JP, Callebaut I. Order in Disorder as Observed by the "Hydrophobic Cluster Analysis" of Protein Sequences. *Proteomics*. 2018;18(21-22):e1800054.

869 36. Callebaut I, Labesse G, Durand P, et al. Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and perspectives. *Cell Mol Life Sci.* 1997;53(8):621-645.

872 37. Bruley A, Bitard-Feildel T, Callebaut I, Duprat E. A sequence-based foldability score combined with AlphaFold2 predictions to disentangle the protein order/disorder continuum. *Proteins*. 2023;91(4):466-484.

875 38. Bruley A, Mornon JP, Duprat E, Callebaut I. Digging into the 3D Structure Predictions of AlphaFold2 with Low Confidence: Disorder and Beyond. *Biomolecules*. 2022;12(10).

878 39. Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. *Nucleic Acids Res.* 2011;39 part (Web Server issue):W29-37.

880 40. Larralde M, Zeller G. PyHMMER: a Python library binding to HMMER for efficient sequence analysis. *Bioinformatics*. 2023;39(5).
882 41. Steinegger M, Söding J. MMseqs2 enables sensitive protein sequence searching for the
883 analysis of massive data sets. *Nat Biotechnol.* 2017;35(11):1026-1028.

884 42. Gibbons TR, Mount SM, Cooper ED, Delwiche CF. Evaluation of BLAST-based edge-
885 weighting metrics used for homology inference with the Markov Clustering algorithm.
886 *BMC Bioinformatics*. 2015;16:218.

887 43. Hagberg A, Swart P, S Chult D. Exploring network structure, dynamics, and function
888 using networkx. Conference: SCIPY 08 ; August 21, 2008 ; Pasadena; 2008; United
889 States.

890 44. Blondel VD, Guillaume J-L, Lambiotte R, Lefebvre E. Fast unfolding of communities
891 in large networks. *Journal of Statistical Mechanics: Theory and Experiment.*
892 2008;2008(10):P10008.

893 45. Shannon P, Markiel A, Ozier O, et al. Cytoscape: a software environment for integrated
894 models of biomolecular interaction networks. *Genome Res.* 2003;13(11):2498-2504.

895 46. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7:
896 improvements in performance and usability. *Mol Biol Evol.* 2013;30(4):772-780.

897 47. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2--a
898 multiple sequence alignment editor and analysis workbench. *Bioinformatics.*
899 2009;25(9):1189-1191.

900 48. Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator.
901 *Genome Res.* 2004;14(6):1188-1190.

902 49. Pettersen EF, Goddard TD, Huang CC, et al. UCSF ChimeraX: Structure visualization
903 for researchers, educators, and developers. *Protein Sci.* 2021;30(1):70-82.

904 50. Tunyasuvunakool K, Adler J, Wu Z, et al. Highly accurate protein structure prediction
905 for the human proteome. *Nature*. 2021;596(7873):590-596.

906 51. van Kempen M, Kim SS, Tumescheit C, et al. Fast and accurate protein structure search
907 with Foldseek. *Nat Biotechnol.* 2023.

908 52. Robert X, Gouet P. Deciphering key features in protein structures with the new
909 ENDscript server. *Nucleic Acids Res.* 2014;42(Web Server issue):W320-324.

910 53. Andersson M, Mattle D, Sitsel O, et al. Copper-transporting P-type ATPases use a
911 unique ion-release pathway. *Nat Struct Mol Biol.* 2014;21(1):43-48.

912 54. Gourdon P, Liu XY, Skjørringe T, et al. Crystal structure of a copper-transporting PIB-
913 type ATPase. *Nature*. 2011;475(7354):59-64.

914 55. Olesen C, Picard M, Winther AM, et al. The structural basis of calcium transport by the
915 calcium pump. *Nature*. 2007;450(7172):1036-1042.

916 56. Toyoshima C, Nakasako M, Nomura H, Ogawa H. Crystal structure of the calcium
917 pump of sarcoplasmic reticulum at 2.6 Å resolution. *Nature*. 2000;405(6787):647-655.

918 57. Smith AT, Smith KP, Rosenzweig AC. Diversity of the metal-transporting P1B-type
919 ATPases. *J Biol Inorg Chem.* 2014;19(6):947-960.

920 58. Thomas C, Aller SG, Beis K, et al. Structural and functional diversity calls for a new
921 classification of ABC transporters. *FEBS Lett.* 2020;594(23):3767-3775.

922 59. Leskelä S, Kontinen VP, Sarvas M. Molecular analysis of an operon in Bacillus subtilis
923 encoding a novel ABC transporter with a role in exoprotein production, sporulation and
924 competence. *Microbiology (Reading)*. 1996;142 (Pt 1):71-77.

925 60. Sigal YJ, McDermott MI, Morris AJ. Integral membrane lipid
926 phosphatases/phosphotransferases: common structure and diverse functions. *Biochem
927 J.* 2005;387(Pt 2):281-293.

928 61. Stukey J, Carman GM. Identification of a novel phosphatase sequence motif. *Protein
929 Sci.* 1997;6(2):469-472.
930 62. Dillon DA, Wu WI, Riedel B, Wissing JB, Dowhan W, Carman GM. The Escherichia coli pgpB gene encodes for a diacylglycerol pyrophosphate phosphatase activity. *J Biol Chem.* 1996;271(48):30548-30553.

933 63. Zhao J, An J, Hwang D, et al. The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis. *mBio*. 2019;10(3).

935 64. Miller DJ, Jerga A, Rock CO, White SW. Analysis of the Staphylococcus aureus DgkB structure reveals a common catalytic mechanism for the soluble diacylglycerol kinases. *Structure*. 2008;16(7):1036-1046.

938 65. Bakali MA, Nordlund P, Hallberg BM. Expression, purification, crystallization and preliminary diffraction studies of the mammalian DAG kinase homologue YegS from Escherichia coli. *Acta Crystallogr Sect F Struct Biol Cryst Commun.* 2006;62(Pt 3):295-297.

942 66. Borgstahl GE, Parge HE, Hickey MJ, Beyer WF, Jr., Hallewell RA, Tainer JA. The structure of human mitochondrial manganese superoxide dismutase reveals a novel tetrameric interface of two 4-helix bundles. *Cell*. 1992;71(1):107-118.

945 67. Kleiger G, Grothe R, Mallick P, Eisenberg D. GXXXG and AXXXA: common alpha-helical interaction motifs in proteins, particularly in extremophiles. *Biochemistry*. 2002;41(19):5990-5997.

948 68. Koehler Leman J, Szczerbiak P, Renfrew PD, et al. Sequence-structure-function relationships in the microbial protein universe. *Nat Commun.* 2023;14(1):2351.

950 69. Jordan IK, Natale DA, Koonin EV, Galperin MY. Independent evolution of heavy metal-associated domains in copper chaperones and copper-transporting atpases. *J Mol Evol.* 2001;53(6):622-633.

953 70. Wang K, Sitsel O, Meloni G, et al. Structure and mechanism of Zn2+-transporting P-type ATPases. *Nature*. 2014;514(7523):518-522.

955 71. Mattle D, Sitsel O, Autzen HE, Meloni G, Gourdon P, Nissen P. On allosteric modulation of P-type Cu(+)-ATPases. *J Mol Biol.* 2013;425(13):2299-2308.

957 72. Biemans-Oldehinkel E, Doeven MK, Poolman B. ABC transporter architecture and regulatory roles of accessory domains. *FEBS Lett.* 2006;580(4):1023-1035.

959 73. Sikkema HR, van den Noort M, Rheinberger J, et al. Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA. *Sci Adv.* 2020;6(47).

961 74. Hwang TC, Braakman I, van der Sluijs P, Callebaut I. Structure basis of CFTR folding, function and pharmacology. *J Cyst Fibros.* 2023;22 Suppl 1:S5-S11.

963 75. Coleman JA, Quazi F, Molday RS. Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport. *Biochim Biophys Acta.* 2013;1831(3):555-574.

965 76. Lyons JA, Timcenko M, Dieudonné T, Lenoir G, Nissen P. P4-ATPases: how an old dog learnt new tricks - structure and mechanism of lipid flippases. *Curr Opin Struct Biol.* 2020;63:65-73.

968 77. Corbalan-Garcia S, Gómez-Fernández JC. Signaling through C2 domains: more than one lipid target. *Biochim Biophys Acta.* 2014;1838(6):1536-1547.

970 78. Lemmon MA. Membrane recognition by phospholipid-binding domains. *Nat Rev Mol Cell Biol.* 2008;9(2):99-111.

972 79. Malcova I, Bumba L, Uljanic F, Kuzmenko D, Nedomova J, Kamanova J. Lipid binding by the N-terminal motif mediates plasma membrane localization of Bordetella effector protein BteA. *J Biol Chem.* 2021;296:100607.

975 80. Varela-Chavez C, Blondel A, Popoff MR. Bacterial intracellularly active toxins: Membrane localisation of the active domain. *Cell Microbiol.* 2020;22(7):e13213.

977 81. López-Lara IM, Geiger O. Bacterial lipid diversity. *Biochim Biophys Acta Mol Cell Biol Lipids.* 2017;1862(11):1287-1299.
979 82. Petroutsos D, Amiar S, Abida H, et al. Evolution of galactoglycerolipid biosynthetic pathways--from cyanobacteria to primary plastids and from primary to secondary plastids. *Prog Lipid Res.* 2014;54:68-85.

980
981
982 83. Wada H, Murata N. Membrane lipids in cyanobacteria. In: Siegenthaler P-A, Murata N, eds. *Lipids in photosynthesis*. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1998:65-81.

985 84. Boudière L, Michaud M, Petroutsos D, et al. Glycerolipids in photosynthesis: composition, synthesis and trafficking. *Biochim Biophys Acta*. 2014;1837(4):470-480.

986
987 85. Wada H, Murata N. The essential role of phosphatidylglycerol in photosynthesis. *Photosynth Res.* 2007;92(2):205-215.

989 86. Kóbori TO, Uzumaki T, Kis M, et al. Phosphatidylglycerol is implicated in divisome formation and metabolic processes of cyanobacteria. *J Plant Physiol.* 2018;223:96-104.

990
991 87. Terrapon N, Weiner J, Grath S, Moore AD, Bornberg-Bauer E. Rapid similarity search of proteins using alignments of domain arrangements. *Bioinformatics*. 2014;30(2):274-281.

994 88. Buchan DWA, Jones DT. Learning a functional grammar of protein domains using natural language word embedding techniques. *Proteins*. 2020;88(4):616-624.
<figure><img src="image_66.png" /><figcaption>**Figure 1:** Topology diagrams and ribbon representations of the 3D structures of (A) an HMA domain (Human CopZ; experimental 3D structure - PDB 2QIF); (B) a CoBaHMA domain (*S. calcipolaris* calcyanin; AlphaFold2 model). Regular secondary structures are colored rainbow, from the N-terminus (blue) to the C-terminus (red), with the exception of the additional strand β0, specific of the CoBaHMA domain (pink). Conserved amino acids, specific to the two families, are highlighted in a ball-and-stick representation.</figcaption></figure>
<figure><img src="image_67.png" /><figcaption>**Figure 2:** Workflow of the current analysis. (A) The 15 sequences of CoBaHMA domains were used as probes for sequence similarity searches with HHBlits against the Uniclust30 database. The 3D structure models of the sequences that were identified by HHBlits were considered in order to assess the presence of strand β0, a feature allowing to distinguish true CoBaHMA domains. The new oBaHMA domain sequences were then used as additional probes with HHBlits to perform transitive searches. (B) Full length proteins with at least one CoBaHMA domain were annotated relative to transmembrane regions, calcyanin signature, structural domains and domains families using deepTMHMM, PCALF, DomainMapper and InterProScan, respectively. In addition, the taxonomy of all sequences is retrieved through the UniProt API. (C) Sequences were clustered using mmseqs2 and representative sequences were searched against themselves. Resulting alignment scores were used to build a similarity network. The network was refined iteratively to ensure that all sequence within a community share a similar length with a maximal amplitude of 50 residues. Finally, nodes and edges were rendered using the edge-rendering and weighted spring embedded layout from cytoscape.</figcaption></figure>
<figure><img src="image_68.png" /><figcaption>**Figure 3:** Logo of the amino acid conservation in the CoBaHMA family sequences. Amino acids are colored according to their properties (Black: Apolar, Green: Polar, Yellow: Small, Orange: Aromatic, Blue: Basic, Red: Acid, Cyan: Histidine). The most conserved polar amino acids are displayed on the CoBaHMA AlphaFold2 3D structure model (UniProt A0A545SE61).</figcaption></figure>
<figure><img src="image_69.png" /><figcaption>**Figure 4:** Taxonomic composition of communities. Each bar corresponds to the taxonomic composition of a community with at least 5 sequences. The number of sequences for phylum with more than 50 sequences is indicated on the bar. The total number of sequences per community is displayed in red on the top of the bar. Phyla with less than 20 sequences are labelled as others. Finally, the community C366 with one sequence belonging to Euryarcheota phylum is marked with an asterisk.</figcaption></figure>
<figure><img src="image_70.png" /><figcaption>**Figure 5:** Modular organization of the full-length CoBaHMA domain proteins. Each line illustrates the representative sequence of a large community (the vertical order follows the horizontal order in Figure 4). Line labels represent the accession of the representative sequence, the community number and the dominant phylum. Functional annotations are indicated along the sequence by shaded areas. Each functional category of domain is highlighted by the following color code: CoBaHMA (green), HMA_2 (lime), HMA (yellow), P-type (cyan) and Serca (darkblue), ABC (orange), Calcyanin Gly-Zip (red), PAP2 (magenta). Membrane regions as identified by deepTMHMM are indicated by gray areas.</figcaption></figure>
<figure><img src="image_71.png" /><figcaption>Figure 6: Single CoBaHMA proteins.</figcaption></figure>

AF2 3D structure models of the representative proteins from the eight communities of single CoBaHMA domain proteins (ribbon representations), colored according to the pLDDT values. The conserved amino acids are shown as ball-and-sticks. Core α-helices and β-strands are only labeled for the first communities, with the exception of C399 (panel E), in which the extra-N-terminal helix takes the place of helix α2. Proteins are referenced with their UniProt accession numbers: A) C437: A0A1N7C237, B) C201: A0A2D3VWG2, C) C583: A0A231P312, D) C675: W1SB97, E) C399:F5RIY2, F) C397: A0A426D834, G) C71: A0A109SXH9, H) C685: A0A371IS94. The multiple sequence alignments of the communities, together with the AF2 predicted secondary structures are provided in Supplementary Data 3.
<figure><img src="image_72.png" /><figcaption>**Figure 7: P-type ATPases.** AF2 3D structure models of the representative proteins from six P-type ATPase communities (ribbon representations), colored according to modular organization (top). Domains are designated as in {Palmgren, 2011 #11}. At the bottom are shown the CoBaHMA domains and the M4 transmembrane helices, colored according to the pLDDT values and with the conserved amino acids shown as ball-and-sticks. Proteins are referenced with their UniProt accession numbers: A) C366: A0A3P1Y6T0 (in the dotted box is shown the M4 3D structure AF2 model of D8F5K4 (C429, full-length 3D structure not shown), B) C525: A0A5C7KBI4, C) C140: A0A1Y6CVZ1, D) C20: A0A3S1CNK6, E) C413: R1CS51, F) C556: A0A7C4R520. C429 (A0A7I7MP94) is not shown, as it shares the same domain architecture as C366 (S-P-C motif in M4). The multiple sequence alignments of the communities, together with the AF2 predicted secondary structures are provided in Supplementary Data 3.</figcaption></figure>
<figure><img src="image_73.png" /><figcaption>**Figure 8:** ABC exporters - EcsC proteins - PAP2 - DAGK. AF2 3D structure models of the representative proteins from communities including ABC exporters, EcsC proteins, type 2 phosphatidylglycerol phosphatases (PAP2) and diacylglycerol kinases (DAGK), colored according to the pLDDT values. CoBaHMA domains are shown at the bottom, with conserved amino acids shown as ball-and-sticks. Proteins are referenced with their UniProt accession numbers: A) C538: A0A2W6BRH6, B) C663: A0A552EVV8, C) C419: A0A3S1IWR7, D) C515: A0A0S3UCN3, E) C553: A0A841V906). The 3D structures of the ABC exporter dimer model (A) was built after superimposition of the AF2 model single chain on the experimental 3D structure of TM287/TM288 (best hit in a HH-PRED search, respecting the distance between the two swapped TMs and the TMD core). The multiple sequence alignments of the communities, together with the AF2 predicted secondary structures are provided in Supplementary Data 3.</figcaption></figure>
<figure><img src="image_74.png" /><figcaption>E C352</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>H C678</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>B C202</figcaption></figure>

<figure><img src="image_77.png" /><figcaption>I C106</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>C C331</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>F C283</figcaption></figure>

<figure><img src="image_80.png" /><figcaption>J C578</figcaption></figure>

<figure><img src="image_81.png" /><figcaption>D C341</figcaption></figure>

<figure><img src="image_82.png" /><figcaption>G C192</figcaption></figure>

<figure><img src="image_83.png" /><figcaption>K C487</figcaption></figure>

<figure><img src="image_84.png" /></figure>

**Figure 9: CoBaHMA domains associated with helical hairpins.** AF2 3D structure models of the representative proteins from communities of CoBaHMA domains associated with helical hairpins (ribbon representations), colored according to the pLDDT values. CoBaHMA domains are shown in the same orientation, with those matching the HMA-2 N-ter profile (Pfam 19991) highlighted with a label. Other profile-matching domains (HMA-2 C-ter, GlyZip) are also labeled. Proteins are referenced with their UniProt accession numbers: A) C393: A0A1Z4FYZ0, B) C202: A0A5C7T941, C) C331: Q8YVH2, D) C341: A0A6M0J010, E) C352: A0A7Y4FMF1, F) C283: B1WT30, G) C192: Q8DMV2, H) C678: A0A7W8FDY5, I) C106: A0A7C2VAZ9, J: C578: A0A1H4BM11, K) C487: H8GQW7. The multiple sequence alignments of the communities, together with the AF2 predicted secondary structures is provided in Supplementary Data 3.
<figure><img src="image_85.png" /><figcaption>**Figure 10: CoBaHMA domains with unknown regions.** AF2 3D structure models of the representative proteins from communities of CoBaHMA domains associated with unknown regions (ribbon representations), colored according to the pLDDT values. Proteins are referenced with their UniProt accession numbers: A) C233: U2SKL7, the CoBaHMA domain is followed by an helical bundle, which superimposed with the MA/MB/M1/M2 bundle of P<sub>IB</sub>-type ATPases (left), B) C4: A0A1H0C0W7, the CoBaHMA domain is followed by an helical bundle, with no striking similarities with any known 3D structures, C) C93: A0A552LCK1, the CoBaHMA domain is preceded by an all-alpha domain, with no striking similarities with any known 3D structures, D) C654: A0A0M0SSI8, three CoBaHMA domains are separated from each other by disordered linkers. The multiple sequence alignments of the communities, together with the AF2 predicted secondary structures is provided in Supplementary Data 3.</figcaption></figure>
Supplementary Table 1:

A. Annotations in the 48 most populated communities (n≥5) using domainMapper and InterProscan. The numbers of sequences which are detected are indicated, with * corresponding to an annotation of the representative sequences of the communities.

<table><thead><tr><th colspan="2">E-value &lt; 1.0e-10</th><th>C20<br>(n=7)</th><th>C556<br>(n=22)</th><th>C413</th><th>C140<br>(n=18)</th><th>C366<br>(n=115)</th><th>C525<br>(n=36)</th><th>C444<br>(n=100)</th><th>C520<br>(n=6)</th><th>C538<br>(n=11)</th><th>C419<br>(n=22)</th></tr></thead><tbody><tr><td rowspan="10">DomainMapper</td><td>2006.1.1.5</td><td>HAD-like</td><td></td><td>C655<br>(n=1)</td><td></td><td></td><td></td><td>C566<br>(n=1)</td><td></td><td></td><td></td></tr><tr><td>5073.1.1.14</td><td>Calcium ATPase transmembrane domain M</td><td></td><td>C430<br>(n=1)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>267.1.1.3</td><td>Metal cation-transporting ATPase, ATP-binding domain</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>10.13.1.2</td><td>Calcium ATPase, transduction domain A</td><td>7*</td><td>23*</td><td>5</td><td>18*</td><td>115*</td><td>28*</td><td></td><td></td><td></td></tr><tr><td>2006.1.1.42</td><td>HAD-like</td><td>3*</td><td>21*</td><td>11*</td><td>18*</td><td>115*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>267.1.1.5</td><td>Metal cation-transporting ATPase, ATP-binding domain</td><td></td><td></td><td>10*</td><td>18*</td><td>115*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>5073.1.2.3</td><td>Copper efflux ATPase transmembrane domain</td><td></td><td></td><td></td><td></td><td>79*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>304.3.1.7</td><td>HMA, heavy metal-associated domain</td><td></td><td></td><td></td><td></td><td></td><td>35*</td><td>4</td><td></td><td></td></tr><tr><td>1075.4.1.1</td><td>Type I ABC exporter transmembrane domain fold</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>6*</td><td>11*</td><td></td></tr><tr><td>2004.1.1.1405</td><td>P-loop containing nucleoside triphosphate hydrolases</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>6*</td><td>11*</td><td></td></tr><tr><td>180.1.1.1</td><td>Acid phosphatase/Vanadium-dependent haloperoxidase</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>23*</td></tr><tr><td rowspan="10">Interproscan</td><td>IPR006068</td><td>Cation-transporting P-type ATPase, C-terminal</td><td></td><td>23*</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IPR004014</td><td>Cation-transporting P-type ATPase, N-terminal</td><td></td><td>23*</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IPR023298</td><td>P-type ATPase, transmembrane domain superfamily</td><td></td><td>24*</td><td></td><td></td><td>2</td><td>29*</td><td></td><td></td><td></td></tr><tr><td>IPR027256</td><td>P-type ATPase, subfamily IB</td><td></td><td></td><td></td><td>18*</td><td>113*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>IPR023299</td><td>P-type ATPase, cytoplasmic domain N</td><td></td><td>24*</td><td>11*</td><td>18*</td><td>115*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>IPR001757</td><td>P-type ATPase</td><td></td><td>24*</td><td>11*</td><td>18*</td><td>115*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>IPR023214</td><td>HAD superfamily</td><td>4*</td><td>24*</td><td>11*</td><td>18*</td><td>115*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>IPR036412</td><td>HAD-like superfamily</td><td>2</td><td>24*</td><td>11*</td><td>18*</td><td>115*</td><td>37*</td><td></td><td></td><td></td></tr><tr><td>IPR044492</td><td>P-type ATPase, haloacid dehalogenase domain</td><td></td><td>24*</td><td>5</td><td>17*</td><td>108*</td><td>35*</td><td></td><td></td><td></td></tr><tr><td>IPR008250</td><td>P-type ATPase, A domain superfamily</td><td></td><td>7*</td><td>24*</td><td>4</td><td>18*</td><td>115*</td><td>37*</td><td></td><td></td></tr><tr><td>IPR036163</td><td>Heavy metal-associated domain superfamily</td><td></td><td></td><td></td><td></td><td></td><td>36*</td><td></td><td></td><td></td></tr><tr><td>IPR006121</td><td>Heavy metal-associated domain, HMA</td><td></td><td></td><td></td><td></td><td></td><td>12*</td><td></td><td></td><td></td></tr><tr><td>IPR000326</td><td>Phosphatidic acid phosphatase type 2/haloperoxidase</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>23*</td></tr><tr><td>IPR036938</td><td>Phosphatidic acid phosphatase type 2/haloperoxidase superfamily</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>23*</td></tr><tr><td rowspan="4">ATP-binding</td><td>IPR039421</td><td>Type 1 protein exporter</td><td></td><td></td><td></td><td></td><td></td><td></td><td>5*</td><td>11*</td><td></td></tr><tr><td>IPR027417</td><td>P-loop containing nucleoside triphosphate hydrolase</td><td></td><td></td><td></td><td></td><td></td><td></td><td>6*</td><td>11*</td><td></td></tr><tr><td>IPR003439</td><td>ABC transporter-like, ATP-binding domain</td><td></td><td></td><td></td><td></td><td></td><td></td><td>6*</td><td>11*</td><td></td></tr><tr><td>IPR003593</td><td>AAA+ ATPase domain</td><td></td><td></td><td></td><td></td><td></td><td></td><td>6*</td><td>11*</td><td></td></tr><tr><td rowspan="2">TM</td><td>IPR036640</td><td>ABC transporter type 1, transmembrane domain superfamily</td><td></td><td></td><td></td><td></td><td></td><td></td><td>6*</td><td>11*</td><td></td></tr><tr><td>IPR011527</td><td>ABC transporter type 1, transmembrane domain</td><td></td><td></td><td></td><td></td><td></td><td></td><td>6*</td><td>11*</td><td></td></tr></tbody></table>
<figure><img src="image_86.png" /><figcaption>B. InterProscan annotations in the 317 small communities (n<5). DUF stands for Domain of Unknown Function.</figcaption></figure>
<figure><img src="image_87.png" /></figure>

**Supplementary Data 1:** pCALF workflow, a tool to annotate calcyanins. First, HMMs profiles specific of the glycine zipper triplication were searched against a set of proteins using pyHMMER. Then, a set of domains known to be related to calcyanins (Y-type, Z-type, X-type and CoBaHMA) were searched using blastp. Proteins with a complete glycine zipper triplication were classified as calcyanins and their features were respectively re added to MSAs and HMMs.
<figure><img src="image_88.png" /></figure>

**Supplementary Data 2:** Number of sequences by phylum recruited during the transitive search. Phyla represented by less than 20 sequences are labeled as others. Sequences from the Chordata and Euryarcheaota phyla were found during the first transitive search. For each search, the total number of sequences is indicated in red.
**Supplementary Data 3:**

Multiple sequence alignments (MSA) of the sequences of the most populated communities, build using MAFFT and rendered using EspPript3. The first two lines report the 2D structures of the AF2 model of the representative sequence of the community, and its amino acid sequence. The initial MSA ( fasta format) is also provided, together with the coordinates of the 3D structure model (pdb format) and the HCA plot (postscript file) of the representative sequence of the considered community.

https://dropsu.sorbonne-universite.fr/s/PatmeKee7GGbkgP

**Supplementary Data 4:**

Annotations by communities: Modular organization of the full-length CoBaHMA domain proteins within the 48 large community (Functional annotations are indicated along the sequence by shaded areas. Each functional category of domain is highlighted by the following color code: CoBaHMA (green), HMA_2 (lime), HMA (yellow), P-type (cyan) and Serca (darkblue), ABC (orange), Calcyanin Gly-Zip (red), PAP2 (magenta). Membrane regions as identified by deepTMHMM are indicated by gray areas.

https://dropsu.sorbonne-universite.fr/s/J7kMXsFY44TpCBK
<figure><img src="image_89.png" /><figcaption>**Supplementary Data 5:** Alluvial plot of the distribution of sequences belonging to large communities from their great functional group to their taxonomy. There are three sets of nodes from left to right corresponding respectively to: great functional groups, communities (with at least 5 sequences) and taxonomy (at the phylum level). The height of a node represents the total number of sequences in the considered category. The width of a link represents the number of sequences belonging to the starting node and the ending node. The color of links between great functional groups and communities and links between communities and phyla correspond to the colors defined in Figure 5 and 4/6, respectively. Communities associated with Single CoBaHMA proteins are marked with an asterisk.</figcaption></figure>
<figure><img src="image_90.png" /></figure>

**Supplementary Data 6:** Similarity network of proteins with at least one CoBaHMA domain. Nodes are full length proteins and edges represent significative relationship. The edge-weighted spring-embedded layout was used with the normalized bitscore as weight to render the network. Thus, similar nodes and highly connected nodes are close to each other. Sub-networks are grouped based on the great functional groups defined in 3.2.2 (CoBaHMA in multidomain proteins): A) Single-CoBaHMA or Non-annotated communities, the insert shows a schematic view of community relationships within the sub-network, B) P-type ATPases, C) ABC transporters, D) Calcyanins, E) PAP2, F) Two CoBaHMA domains proteins, G) Three CoBaHMA domains proteins.
<figure><img src="image_91.png" /></figure>

**Supplementary Data 7:**

HCA plot of the representative sequence of the C352 community (UniProt A0A7Y4FMF1). The sequence is shown on an duplicated α-helical net, on which strong hydrophobic amino acids (V,I,L,M,F,Y,W) are contoured. They form hydrophobic clusters, which have been shown to mainly correspond to regular secondary structures. Symbols and the way to read the sequence and secondary structures are indicated in the inset. Although the C-terminus is predicted as disordered on the AF2 model, it contains large hydrophobic clusters, which are indicative of the presence of regular secondary structures.

<figure><img src="image_92.png" /></figure>

**Supplementary Data 8:**

Example of the interaction of a CoBaHMA domain with another domain of a PIB-type ATPase (community C140, UniProt A0A1U7CQU6). The inset illustrates the bond network involving two amino acids of the CoBaHMA domain
**Abstract :**

Biomineralisation is all the processes that lead to the formation of minerals by living beings. In 2012, a new biomineralization phenotype has been described in cyanobacteria, characterized by the presence of amorphous alkaline-earth carbonate inclusions inside the cells.

A comparative genomic analysis revealed that this intracellular biomineralisation phenotype is linked to the presence of one gene, unknown at the time, which has been called *ccyA*. It codes for one protein called calcyanin. Calcyanin has 4 variants, that share the same C-terminus domain ((GlyZip)₃), but which differ in their N-terminus domain (CoBaHMA, X, Y or Z). None of these 5 domains has already been described in the literature.

The goal of this PhD was to characterize the 3D structure of *Synechococcus calcipolaris*'s calcyanin, which has a CoBaHMA domain, by combining bioinformatics and experimental approaches, in order to make hypothesis regarding its role.

Through sequence analysis and 3D structure modeling, we showed that the CoBaHMA domain belongs to the “ferredoxin-like” fold, typical of the superfamily HMA (“Heavy Metal Associated”), and sets itself as a new family in it, characterized by conserved basic amino acids and an additional β strand (1). We have performed sequence similarity searches, refined with the structural information of the 3D structure models. This way, we showed that the CoBaHMA domain can be found on several different protein architectures, in various taxa. It has an independent domain, or in conjunction with other domains, especially membrane systems which, among others, allow transports of substrates through the membrane (P<sub>IB</sub>-type ATPases, ABC exporters) or new families with unknown functions. These results lead us to formulate hypotheses regarding the CoBaHMA domain function (2).

We also proposed a robust model for the individual glycine zipper from which the name of the C-terminus domain (GlyZip)₃ of calcyanins comes from. These glycine zippers have a structure of a compact hairpin made of two helices, which is akin to the ones of transmembrane proteins that form pore. However, we were not able to model satisfyingly their assembly nor their possible interactions with the CoBaHMA domain, emphasizing the importance of studying the protein experimentally.

We successfully expressed calcyanin in *Escherichia coli*, and purified it. However the protein proved to be quite unstable, with a propensity to form a great diversity of objects with different sizes. A limited proteolysis experiment revealed the existence of a protease-resistant fragment of calcyanin, which encompasses the CoBaHMA domain and the first glycine zipper of the (GlyZip)₃ domain. We expressed and purified this fragment, fused to MBP (« Maltose Binding Protein »). The fragment forms only one object in solution, but is prone to precipitation once separated from MBP. Yet we have successfully obtained crystals of this fragment, which pave the way to solve its experimental 3D structure.

Calcyanin is a difficult protein to work with, both experimentally and by bioinformatics. But we managed to model and characterize several of its fragments. From that, we inferred relevant information on calcyanin. More specifically, we highlighted a new family of domains, CoBaHMA, which presence on other protein architectures opens up new hints to understand its function and evolution.

<footnote>(1) Benzerara *et al.*, 2022 14(3): evav026. doi:10.1093/gbe/evac026.</footnote>

<footnote>(2) Gaschignard *et al.*, En préparation.</footnote>
Résumé :

La biominéralisation est l'ensemble des processus qui aboutissent à la formation de minéraux par des êtres vivants. En 2012, un nouveau phénotype de biominéralisation a été décrit chez les cyanobactéries, caractérisé par la présence de précipités amorphes de carbonate d'alcalino-terreux au sein des cellules. Une analyse de génomique comparative a montré que ce phénotype de biominéralisation intracellulaire était associé à la présence d'un gène, encore non caractérisé, qui a été nommé ccyA, et qui code pour une protéine nommée calcyanine. La calcyanine a 4 variants qui partagent un même domaine C-terminal ((GlyZip)₃), mais qui diffèrent au niveau de leur domaine N-terminal (CoBaHMA, X, Y ou Z). Aucun de ces 5 domaines n'est encore décrit dans la littérature.

L'objectif de cette thèse a été de caractériser la structure 3D de la calcyanine de *Synechococcus calcipolaris*, variant à domaine CoBaHMA, en combinant des approches bioinformatiques et expérimentales, afin de progresser dans la connaissance de sa fonction.

En combinant un ensemble de méthodes d'analyse de séquences et de modélisation de 3D structures, nous avons montré que le domaine CoBaHMA adopte un repliement « ferredoxin-like » typique de la superfamille HMA (« Heavy-Metal Associated ») et forme au sein de celle-ci une nouvelle famille, caractérisée par la conservation d'acides aminés basiques et la présence d'un brin β additionnel (1). Nous avons ensuite réalisé des recherches avancées de similitudes de séquence, intégrant les informations dérivées des modèles de structure 3D. Nous avons ainsi montré que le domaine CoBaHMA est présent dans différentes organisations modulaires au sein de taxa variés. Il existe sous la forme de domaine unique, ou accompagné d'autres domaines, en particulier des systèmes membranaires qui permettent, entre autre, le transport de substrats au travers des membranes (P<sub>IB</sub>-type ATPases, exporteurs ABC) ou qui constituent de nouvelles familles aux fonctions encore inconnues. Ces résultats permettent de proposer des hypothèses quant à la fonction moléculaire du domaine CoBaHMA et aux processus cellulaires dans lesquels il est impliqué (2).

Nous avons également proposé un modèle cohérent des trois motifs glycine zippers individuels qui donnent leur nom au domaine (GlyZip)₃ C-terminal des calcyanines. Ces glycine zippers forment une structure compacte de 2 hélices en épingle à cheveux, qui rappelle celle adoptée par des protéine transmembranaires formant des pores. Cependant, nous n'avons pu obtenir de modèle satisfaisant de l'assemblage des motifs glycine zipper (domaine (GlyZip)₃), ni de leur interaction potentielle avec le domaine CoBaHMA, renforçant ainsi l'intérêt d'une étude expérimentale.

Nous avons ainsi exprimé la calcyanine de *S.calcipolaris* chez *Escherichia coli*, et l'avons purifiée. Cependant cette protéine s'est avérée très peu stable, avec une propension à former un grand nombre d'objets de taille différentes en solution. Une expérience de protéolyse limitée a montré l'existence d'un fragment de la calcyanine résistant aux protéases, constitué du domaine CoBaHMA et du premier glycine zipper du domaine (GlyZip)₃. Nous avons donc exprimé et purifié ce fragment fusionné à la protéine MBP (« Maltose Binding Protein »), afin d'en augmenter la solubilité. Celui-ci ne forme qu'une espèce en solution, mais est très peu soluble après avoir été séparé de la MBP. Nous avons cependant réussi à obtenir des cristaux de ce fragment offrant des perspectives encourageantes pour résoudre sa structure expérimentale.

La calcyanine est donc une protéine difficile à étudier, tant par voie expérimentale que par
modélisation. Néanmoins nous avons réussi à modéliser et caractériser plusieurs de ses fragments.
Nous avons déduit de cette étude des informations pertinentes sur cette protéine, et plus
particulièrement de son domaine CoBaHMA, dont la présence dans d'autres architectures protéiques
ouvre des perspectives pour comprendre sa fonction et son évolution.

<footnote>(1) Benzerara *et al.*, 2022 14(3): evav026. doi:10.1093/gbe/evac026</footnote>

<footnote>(2) Gaschignard et al., En préparation.</footnote>