bioRxiv preprint doi: https://doi.org/10.1101/207324; this version posted October 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Conservation of specificity in two low-specificity 2 proteins 1,2 1,2 1,2 3 Lucas C. Wheeler, Jeremy A. Anderson , Anneliese J. Morrison , Caitlyn E. Wong1,2, Michael J. Harms1,2,* 4 1.Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, 5 USA 6 2. Institute of Molecular Biology, University of Oregon, Eugene, OR, USA 7 * [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/207324; this version posted October 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 8 Abstract 9 S100 proteins bind linear peptide regions of target proteins and modulate their ac- 10 tivity. The peptide binding interface, however, has remarkably low specificity and 11 can interact with many target peptides. It is not clear if the interface discrimi- 12 nates targets in a biological context, or whether biological specificity is achieved 13 exclusively through external factors such as subcellular localization. To discriminate 14 these possibilities, we used an evolutionary biochemical approach to trace the evolu- 15 tion of paralogs S100A5 and S100A6. We first used isothermal titration calorimetry 16 to study the binding of a collection of peptides with diverse sequence, hydrophobic- 17 ity, and charge to human S100A5 and S100A6. These proteins bound distinct, but 18 overlapping, sets of peptide targets. We then studied the peptide binding properties 19 of S100A5 and S100A6 orthologs sampled from across five representative amniote 20 species. We found that the pattern of binding specificity was conserved along all 21 lineages, for the last 320 million years, despite the low specificity of each protein. 22 We next used Ancestral Sequence Reconstruction to determine the binding speci- 23 ficity of the last common ancestor of the paralogs. We found the ancestor bound 24 the whole set of peptides bound by modern S100A5 and S100A6 proteins, suggesting 25 that paralog specificity evolved by subfunctionalization. To rule out the possibility 26 that specificity is conserved because it is difficult to modify, we identified a single 27 historical mutation that, when reverted in human S100A5, gave it the ability to bind 28 an S100A6-specific peptide. These results indicate that there are strong evolutionary 2 bioRxiv preprint doi: https://doi.org/10.1101/207324; this version posted October 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 29 constraints on peptide binding specificity, and that, despite being able to bind a large 30 number of targets, the specificity of S100 peptide interfaces is indeed important for 31 the biology of these proteins. 32 Introduction 33 Many proteins have low specificity interfaces that can interact with a wide variety of 34 targets (1–11). Such interfaces are difficult to dissect. Crucially, it is not obvious that 35 their specificity is biologically meaningful: maybe such proteins are essentially indis- 36 criminate, and biological specificity is encoded by external factors such as subcellular 37 localization or expression pattern (3, 12, 13). 38 An evolutionary perspective allows us to probe whether specificity is, indeed, an 39 important aspect of these interfaces (14). If there are functional and evolutionary 40 constraints on binding partners, we would expect conservation of binding specificity 41 similar to that observed for high-specificity protein families (15, 16). In contrast, if 42 specificity is unimportant, we would expect it to fluctuate randomly over evolution- 43 ary time. Further, previous work on the evolution of specificity has revealed common 44 patterns for the evolution of specificity (17–19), including partitioning of ancestral 45 binding partners among descendant lineages (20–23) and transitions through more 46 promiscuous intermediates (10, 24, 25). If low-specificity proteins exhibit similar 47 patterns, it is strong evidence that the low specificity interface has conserved bind- 48 ing properties, and that the interface makes a meaningful contribution to biological 49 specificity. 50 S100 proteins are an important group of low-specificity proteins (26, 27). Mem- 3 bioRxiv preprint doi: https://doi.org/10.1101/207324; this version posted October 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 51 bers of the family act as metal sensors (28), pro-inflammatory signals (29–32), and 52 antimicrobial peptides (33). Most S100s bind to linear peptide regions of target 2+ 53 proteins via a short hydrophobic interface exposed on Ca -binding (Fig 1A). S100s 54 recognize extremely diverse protein targets (27, 34, 35). No simple sequence motif 55 for discriminating binders from non-binders has yet been defined. The breadth of 56 targets is much more extreme than other low-specificity proteins such as kinases and 57 some hub proteins, which recognize well-defined, but degenerate, sequence motifs 58 (1, 3, 6, 10, 11). 59 We set out to determine whether there was conserved specificity for two S100 60 paralogs, S100A5 and S100A6. These proteins arose by gene duplication in the 61 amniote ancestor ≈ 320 million years ago (36, 37). S100A6 regulates the cell cycle 62 and cellular motility in response to stress (38). It binds to many targets including 63 p53 (39, 40), RAGE (31), Annexin A1 (35), and Siah-interacting protein (41). A 64 crystal structure of human S100A6 bound to a fragment of Siah-interacting protein 65 revealed that peptides bind via the canonical hydrophobic interface shared by most 66 S100 proteins (41). The biology of S100A5 is less well understood. It binds both 67 RAGE (31, 32) and a fragment of the protein NCX1 (42) at the canonical binding 68 site. It is highly expressed in mammalian olfactory tissues (43–45), but its specific 69 targets and their biological roles are not well understood. 70 Using a combination of in vitro biochemistry and molecular phylogenetics, we 71 addressed three key questions regarding the evolution of specificity in S100A5 and 72 S100A6. First: do the two human proteins exhibit specificity relative to one another? 73 Second: is the set of binding partners recognized by each protein fixed over time, 4 bioRxiv preprint doi: https://doi.org/10.1101/207324; this version posted October 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 74 or does the set of partners fluctuate? And, third: do we see similar patterns of 75 specificity change after gene duplication for these low-specificity proteins compared 76 to high-specificity proteins? Unsurprisingly, we find that S100A5 and S100A6 both 77 bind to a wide variety of diverse peptides. Surprisingly, we find that the set of 78 partners, despite being diverse, has been conserved over hundreds of millions of 79 years. Further, we observe a pattern of subfunctionalization for these low-specificity 80 proteins that is identical to that observed in high-specificity proteins. This suggests 81 that these low-specificity interfaces are indeed constrained to maintain a specific—if 82 large—set of binding targets. 83 Results 84 Human S100A5 and S100A6 interact with diverse peptides at the same 85 binding site 86 We first systematically compared the binding specificity of human S100A5 (hA5) 87 relative to human S100A6 (hA6) for a collection of six peptides (Fig 1B). Peptide 88 targets have been reported for both hA5 and hA6 (31, 32, 35, 39–42), but only two 89 targets have been directly compared between paralogs. Using Isothermal Titration 90 Calorimetry (ITC), Streicher and colleagues found that a peptide fragment of An- 91 nexin 1 bound to hA6 but not hA5, and a peptide fragment of Annexin 2 bound to 92 neither (35) (Fig 1B). To better quantify the relative specificity of these proteins, we 93 used ITC to measure the binding of two additional peptides to recombinant hA5 and 94 hA6. The first was a peptide from Siah-interacting protein (SIP) previously reported 95 to bind to hA6 (41). We found that this peptide bound to hA6 with a KD of 20 µM, 5 bioRxiv preprint doi: https://doi.org/10.1101/207324; this version posted October 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 96 but did not bind hA5 (Fig 1B, C). The second was a 12 amino acid fragment of the 97 protein NCX1 that was reported to bind to hA5 (42). We found that this peptide 98 bound with to hA5 with a KD of 20 µM, but did not bind hA6 (Fig 1B, C). 99 To further characterize the specificity of the interface, we used phage display to 100 identify two additional peptides that bound to each protein.