New binding specificities evolve via point mutation in an invertebrate allorecognition gene

Abstract

Many organisms use genetic self-recognition systems to distinguish themselves from conspecifics. In the cnidarian, Hydractinia symbiolongicarpus, self-recognition is partially controlled by allorecognition 2 (Alr2). Alr2 encodes a highly polymorphic transmembrane protein that discriminates self from nonself by binding in trans to other Alr2 proteins with identical or similar sequences. Here, we focused on the N-terminal domain of Alr2, which can determine its binding specificity. We pair ancestral sequence reconstruction and experimental assays to show that amino acid substitutions can create sequences with novel binding specificities either directly (via one mutation) or via sequential mutations and intermediates with relaxed specificities. We also show that one side of the domain has experienced positive selection and likely forms the binding interface. Our results provide direct evidence that point mutations can generate Alr2 proteins with novel binding specificities. This provides a plausible mechanism for the generation and maintenance of functional variation in nature.

Keywords: Evolutionary Biology; Molecular Biology; Molecular Genetics.

Graphical abstract

Figure 1

Isoform-specific, homophilic binding of Alr2 isoforms (A) Alr2 protein structure. SP = Signal peptide, ECS = Extracellular spacer, TM = Transmembrane domain, CT = Cytoplasmic tail. (B) Multiple sequence alignment of 111A06 and 214E06 domain 1. Polymorphisms highlighted in purple. (C) Cell aggregation assays of 111A06 and 214E06. Cells transfected with vectors encoding only fluorescent proteins (eGFP or mRuby2) do not form aggregates (bottom right). Scale bar = 100 μm.

Figure 2

Evolution of novel binding specificities via point mutation (A) Maximum-likelihood tree of 146 domain 1 coding sequences. (B) Expansion of clade that includes 111A06 and 214E06. Allele names on branch. Amino acid changes indicated along branches. (C) Multiple sequence alignment of clade. Variant residues highlighted. (D) Plasmid map Alr2 fusion proteins. (E-H) Representative images of cell aggregation assays. (E) Anc, 046B, and Hap074 against themselves. (F) Anc, 046B, and Hap074 against 111A06. (G) All pairwise combinations of Anc, 046B, and Hap074. (H) Anc, 046B, and Hap074 versus 214E06. Arrowheads point to semi-mixed aggregates (See also Figure S1). Scale bar = 100 μm. (I) Node network of isoforms colored by binding specificity. Triangles indicate the hypothesized direction of mutation from Anc. Green dotted lines indicate weaker heterophilic interactions.

Figure 3

Domain 1 isoforms can evolve via intermediates with broadened specificity (A) Expanded node network including hypothesized single-step mutants between Anc and 046B, Hap074 and 214E06. Scale bar = 100 μm and applies to all images. (B–H) Representative images of cell aggregation assays. (B) Mutants tested against themselves. (C) Anc-T76R and Anc-E93K tested against Anc, 046B, Hap074. (D) Anc-T76R versus 111A06 (left) and Anc-E93K versus 111A06 (right). Semi-mixed aggregates indicated with arrowheads (See also Figure S1A). (E) Anc-T76R and Anc-E93K versus 214E06 (See also Figure S1C). (F) Hap074-S44G and Hap074-G47E versus 111A06. (G) Hap074-S44G and Hap074-G47E versus 214E06. (H) Hap074-S44G and Hap074-G47E versus all remaining isoforms.

Figure 4

Effects of N32Y mutation on binding specificity and structural analysis (A) Results of assays between N32Y mutants and 111A06 (See also Figures S1G and S1H). (B) Binding profiles of 111A06, 046B-N32Y, Hap074-N32Y, 046B, and Hap074. Asterisk denotes the result of an allele and its N32Y mutant. (C) Binding profiles of 214E06 and 214E06-N32Y. (D) Predicted structure of Anc domain 1. Six variant residues labeled. (E) Sequence conservation mapped onto domain 1. (F) Residues predicted to have experienced either diversifying or purifying selection mapped onto domain 1. Colors correspond to the predictions of MEME and/or FEL. Arrowhead indicates the one residue predicted to be under positive selection by FEL only. (G) Hypothetical binding topologies Alr2.