Imperfect symmetry facilitated the evolution of specificity and high-order stoichiometry in vertebrate hemoglobin.

Many proteins form paralogous multimers - molecular complexes in which evolutionarily related proteins are arranged into specific quaternary structures. Little is known about the mechanisms by which they acquired their stoichiometry (the number of total subunits in the complex) and heterospecificity (the preference of subunits for their paralogs rather than other copies of the same protein). Here we use ancestral protein reconstruction and biochemical experiments to study historical increases in stoichiometry and specificity during the evolution of vertebrate hemoglobin (Hb), a α2ß2 heterotetramer that evolved from a homodimeric ancestor after a gene duplication. We show that the mechanisms for this evolutionary transition was simple. One hydrophobic substitution in subunit ß after the gene duplication was sufficient to cause the ancestral dimer to homotetramerize with high affinity across a new interface. During this same interval, a single-residue deletion in subunit α at the older interface conferred specificity for the heterotetrameric form and the trans-orientation of subunits within it. These sudden transitions in stoichiometry and specificity were possible because the interfaces in Hb are isologous - involving the same surface patch on interacting subunits, rotated 180° relative to each other - but the symmetry is slightly imperfect. This architecture amplifies the impacts of individual mutations on stoichiometry and specificity, especially in higher-order complexes, and allows single substitutions to differentially affect heteromeric vs homomeric interactions. Many multimers are isologous, and symmetry in proteins is always imperfect; our findings therefore suggest that elaborate and specific molecular complexes may often evolve via simple genetic and physical mechanisms.

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme.

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assemblies and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

Epistasis facilitates functional evolution in an ancient transcription factor.

A protein's genetic architecture - the set of causal rules by which its sequence produces its functions - also determines its possible evolutionary trajectories. Prior research has proposed that the genetic architecture of proteins is very complex, with pervasive epistatic interactions that constrain evolution and make function difficult to predict from sequence. Most of this work has analyzed only the direct paths between two proteins of interest - excluding the vast majority of possible genotypes and evolutionary trajectories - and has considered only a single protein function, leaving unaddressed the genetic architecture of functional specificity and its impact on the evolution of new functions. Here, we develop a new method based on ordinal logistic regression to directly characterize the global genetic determinants of multiple protein functions from 20-state combinatorial deep mutational scanning (DMS) experiments. We use it to dissect the genetic architecture and evolution of a transcription factor's specificity for DNA, using data from a combinatorial DMS of an ancient steroid hormone receptor's capacity to activate transcription from two biologically relevant DNA elements. We show that the genetic architecture of DNA recognition consists of a dense set of main and pairwise effects that involve virtually every possible amino acid state in the protein-DNA interface, but higher-order epistasis plays only a tiny role. Pairwise interactions enlarge the set of functional sequences and are the primary determinants of specificity for different DNA elements. They also massively expand the number of opportunities for single-residue mutations to switch specificity from one DNA target to another. By bringing variants with different functions close together in sequence space, pairwise epistasis therefore facilitates rather than constrains the evolution of new functions.

The simplicity of protein sequence-function relationships.

How complicated is the genetic architecture of proteins - the set of causal effects by which sequence determines function? High-order epistatic interactions among residues are thought to be pervasive, making a protein's function difficult to predict or understand from its sequence. Most studies, however, used methods that overestimate epistasis, because they analyze genetic architecture relative to a designated reference sequence - causing measurement noise and small local idiosyncrasies to propagate into pervasive high-order interactions - or have not effectively accounted for global nonlinearity in the sequence-function relationship. Here we present a new reference-free method that jointly estimates global nonlinearity and specific epistatic interactions across a protein's entire genotype-phenotype map. This method yields a maximally efficient explanation of a protein's genetic architecture and is more robust than existing methods to measurement noise, partial sampling, and model misspecification. We reanalyze 20 combinatorial mutagenesis experiments from a diverse set of proteins and find that additive and pairwise effects, along with a simple nonlinearity to account for limited dynamic range, explain a median of 96% of total variance in measured phenotypes (and >92% in every case). Only a tiny fraction of genotypes are strongly affected by third- or higher-order epistasis. Genetic architecture is also sparse: the number of terms required to explain the vast majority of variance is smaller than the number of genotypes by many orders of magnitude. The sequence-function relationship in most proteins is therefore far simpler than previously thought, opening the way for new and tractable approaches to characterize it.

Evolutionary Shortcuts via Multinucleotide Substitutions and Their Impact on Natural Selection Analyses.

Inference and interpretation of evolutionary processes, in particular of the types and targets of natural selection affecting coding sequences, are critically influenced by the assumptions built into statistical models and tests. If certain aspects of the substitution process (even when they are not of direct interest) are presumed absent or are modeled with too crude of a simplification, estimates of key model parameters can become biased, often systematically, and lead to poor statistical performance. Previous work established that failing to accommodate multinucleotide (or multihit, MH) substitutions strongly biases dN/dS-based inference towards false-positive inferences of diversifying episodic selection, as does failing to model variation in the rate of synonymous substitution (SRV) among sites. Here, we develop an integrated analytical framework and software tools to simultaneously incorporate these sources of evolutionary complexity into selection analyses. We found that both MH and SRV are ubiquitous in empirical alignments, and incorporating them has a strong effect on whether or not positive selection is detected (1.4-fold reduction) and on the distributions of inferred evolutionary rates. With simulation studies, we show that this effect is not attributable to reduced statistical power caused by using a more complex model. After a detailed examination of 21 benchmark alignments and a new high-resolution analysis showing which parts of the alignment provide support for positive selection, we show that MH substitutions occurring along shorter branches in the tree explain a significant fraction of discrepant results in selection detection. Our results add to the growing body of literature which examines decades-old modeling assumptions (including MH) and finds them to be problematic for comparative genomic data analysis. Because multinucleotide substitutions have a significant impact on natural selection detection even at the level of an entire gene, we recommend that selection analyses of this type consider their inclusion as a matter of routine. To facilitate this procedure, we developed, implemented, and benchmarked a simple and well-performing model testing selection detection framework able to screen an alignment for positive selection with two biologically important confounding processes: site-to-site synonymous rate variation, and multinucleotide instantaneous substitutions.

Simple mechanisms for the evolution of protein complexity.

Proteins are tiny models of biological complexity: specific interactions among their many amino acids cause proteins to fold into elaborate structures, assemble with other proteins into higher-order complexes, and change their functions and structures upon binding other molecules. These complex features are classically thought to evolve via long and gradual trajectories driven by persistent natural selection. But a growing body of evidence from biochemistry, protein engineering, and molecular evolution shows that naturally occurring proteins often exist at or near the genetic edge of multimerization, allostery, and even new folds, so just one or a few mutations can trigger acquisition of these properties. These sudden transitions can occur because many of the physical properties that underlie these features are present in simpler proteins as fortuitous by-products of their architecture. Moreover, complex features of proteins can be encoded by huge arrays of sequences, so they are accessible from many different starting points via many possible paths. Because the bridges to these features are both short and numerous, random chance can join selection as a key factor in explaining the evolution of molecular complexity.

Epistatic drift causes gradual decay of predictability in protein evolution.

Epistatic interactions can make the outcomes of evolution unpredictable, but no comprehensive data are available on the extent and temporal dynamics of changes in the effects of mutations as protein sequences evolve. Here, we use phylogenetic deep mutational scanning to measure the functional effect of every possible amino acid mutation in a series of ancestral and extant steroid receptor DNA binding domains. Across 700 million years of evolution, epistatic interactions caused the effects of most mutations to become decorrelated from their initial effects and their windows of evolutionary accessibility to open and close transiently. Most effects changed gradually and without bias at rates that were largely constant across time, indicating a neutral process caused by many weak epistatic interactions. Our findings show that protein sequences drift inexorably into contingency and unpredictability, but that the process is statistically predictable, given sufficient phylogenetic and experimental data.

Molecular and structural basis of olfactory sensory neuron axon coalescence by Kirrel receptors.

Projections from sensory neurons of olfactory systems coalesce into glomeruli in the brain. The Kirrel receptors are believed to homodimerize via their ectodomains and help separate sensory neuron axons into Kirrel2- or Kirrel3-expressing glomeruli. Here, we present the crystal structures of homodimeric Kirrel receptors and show that the closely related Kirrel2 and Kirrel3 have evolved specific sets of polar and hydrophobic interactions, respectively, disallowing heterodimerization while preserving homodimerization, likely resulting in proper segregation and coalescence of Kirrel-expressing axons into glomeruli. We show that the dimerization interface at the N-terminal immunoglobulin (IG) domains is necessary and sufficient to create homodimers and fail to find evidence for a secondary interaction site in Kirrel ectodomains. Furthermore, we show that abolishing dimerization of Kirrel3 in vivo leads to improper formation of glomeruli in the mouse accessory olfactory bulb as observed in Kirrel3-/- animals. Our results provide evidence for Kirrel3 homodimerization controlling axonal coalescence.

Contingency and chance erase necessity in the experimental evolution of ancestral proteins.

The roles of chance, contingency, and necessity in evolution are unresolved because they have never been assessed in a single system or on timescales relevant to historical evolution. We combined ancestral protein reconstruction and a new continuous evolution technology to mutate and select proteins in the B-cell lymphoma-2 (BCL-2) family to acquire protein-protein interaction specificities that occurred during animal evolution. By replicating evolutionary trajectories from multiple ancestral proteins, we found that contingency generated over long historical timescales steadily erased necessity and overwhelmed chance as the primary cause of acquired sequence variation; trajectories launched from phylogenetically distant proteins yielded virtually no common mutations, even under strong and identical selection pressures. Chance arose because many sets of mutations could alter specificity at any timepoint; contingency arose because historical substitutions changed these sets. Our results suggest that patterns of variation in BCL-2 sequences - and likely other proteins, too - are idiosyncratic products of a particular and unpredictable course of historical events.

One of the most fundamental and unresolved questions in evolutionary biology is whether the outcomes of evolution are predictable. Is the diversity of life we see today the expected result of organisms adapting to their environment throughout history (also known as natural selection) or the product of random chance? Or did chance events early in history shape the paths that evolution could take next, determining the biological forms that emerged under natural selection much later? These questions are hard to study because evolution happened only once, long ago. To overcome this barrier, Xie, Pu, Metzger et al. developed an experimental approach that can evolve reconstructed ancestral proteins that existed deep in the past. Using this method, it is possible to replay evolution multiple times, from various historical starting points, under conditions similar to those that existed long ago. The end products of the evolutionary trajectories can then be compared to determine how predictable evolution actually is. Xie, Pu, Metzger et al. studied proteins belonging to the BCL-2 family, which originated some 800 million years ago. These proteins have diversified greatly over time in both their genetic sequences and their ability to bind to specific partner proteins called co-regulators. Xie, Pu, Metzger et al. synthesized BCL-2 proteins that existed at various times in the past. Each ancestral protein was then allowed to evolve repeatedly under natural selection to acquire the same co-regulator binding functions that evolved during history. At the end of each evolutionary trajectory, the genetic sequence of the resulting BCL-2 proteins was recorded. This revealed that the outcomes of evolution were almost completely unpredictable: trajectories initiated from the same ancestral protein produced proteins with very different sequences, and proteins launched from different ancestral starting points were even more dissimilar. Further experiments identified the mutations in each trajectory that caused changes in coregulator binding. When these mutations were introduced into other ancestral proteins, they did not yield the same change in function. This suggests that early chance events influenced each protein's evolution in an unpredictable way by opening and closing the paths available to it in the future. This research expands our understanding of evolution on a molecular level whilst providing a new experimental approach for studying evolutionary drivers in more detail. The results suggest that BCL-2 proteins, in all their various forms, are unique products of a particular, unpredictable course of history set in motion by ancient chance events.

A hydrophobic ratchet entrenches molecular complexes.

Most proteins assemble into multisubunit complexes1. The persistence of these complexes across evolutionary time is usually explained as the result of natural selection for functional properties that depend on multimerization, such as intersubunit allostery or the capacity to do mechanical work2. In many complexes, however, multimerization does not enable any known function3. An alternative explanation is that multimers could become entrenched if substitutions accumulate that are neutral in multimers but deleterious in monomers; purifying selection would then prevent reversion to the unassembled form, even if assembly per se does not enhance biological function3-7. Here we show that a hydrophobic mutational ratchet systematically entrenches molecular complexes. By applying ancestral protein reconstruction and biochemical assays to the evolution of steroid hormone receptors, we show that an ancient hydrophobic interface, conserved for hundreds of millions of years, is entrenched because exposure of this interface to solvent reduces protein stability and causes aggregation, even though the interface makes no detectable contribution to function. Using structural bioinformatics, we show that a universal mutational propensity drives sites that are buried in multimeric interfaces to accumulate hydrophobic substitutions to levels that are not tolerated in monomers. In a database of hundreds of families of multimers, most show signatures of long-term hydrophobic entrenchment. It is therefore likely that many protein complexes persist because a simple ratchet-like mechanism entrenches them across evolutionary time, even when they are functionally gratuitous.

Comment on "Ancient origins of allosteric activation in a Ser-Thr kinase".

Hadzipasic et al (Reports, 21 February 2020, p. 912) used ancestral sequence reconstruction to identify historical sequence substitutions that putatively caused Aurora kinases to evolve allosteric regulation. We show that their results arise from using an implausible phylogeny and sparse sequence sampling. Addressing either problem reverses their inferences: Allostery and the amino acids that confer it were not gained during the diversification of eukaryotes but were lost in a subgroup of Fungi.

Author Correction: Origin of complexity in haemoglobin evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Origin of complexity in haemoglobin evolution.

Most proteins associate into multimeric complexes with specific architectures1,2, which often have functional properties such as cooperative ligand binding or allosteric regulation3. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and ß-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical 'missing link' through which the modern tetramer evolved-a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and ß-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein's structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.

Fitness effects but no temperature-mediated balancing selection at the polymorphic Adh gene of Drosophila melanogaster.

Polymorphism in the alcohol dehydrogenase (ADH) protein of Drosophila melanogaster, like genetic variation in many other enzymes, has long been hypothesized to be maintained by a selective trade-off between thermostability and enzyme activity. Two major Adh variants, named Fast and Slow, are distributed along latitudinal clines on several continents. The balancing selection trade-off hypothesis posits that Fast is favored at high latitudes because it metabolizes alcohol faster, whereas Slow is favored at low latitudes because it is more stable at high temperatures. Here we use biochemical and physiological assays of precisely engineered genetic variants to directly test this hypothesis. As predicted, the Fast protein has higher catalytic activity than Slow, and both the Fast protein and regulatory variants linked to it confer greater ethanol tolerance on transgenic animals. But we found no evidence of a temperature-mediated trade-off: The Fast protein is not less stable or active at high temperatures, and Fast alleles increase ethanol tolerance and survivorship at all temperatures tested. Further, analysis of a population genomic dataset reveals no signature of balancing selection in the Adh gene. These results provide strong evidence against balancing selection driven by a stability/activity trade-off in Adh, and they justify caution about this hypothesis for other enzymes except those for which it has been directly tested. Our findings tentatively suggest that environment-specific selection for the Fast allele, coupled with demographic history, may have produced the observed pattern of Adh variation.

EvoChromo: towards a synthesis of chromatin biology and evolution.

Over the past few years, interest in chromatin and its evolution has grown. To further advance these interests, we organized a workshop with the support of The Company of Biologists to debate the current state of knowledge regarding the origin and evolution of chromatin. This workshop led to prospective views on the development of a new field of research that we term 'EvoChromo'. In this short Spotlight article, we define the breadth and expected impact of this new area of scientific inquiry on our understanding of both chromatin and evolution.

Family of neural wiring receptors in bilaterians defined by phylogenetic, biochemical, and structural evidence.

The evolution of complex nervous systems was accompanied by the expansion of numerous protein families, including cell-adhesion molecules, surface receptors, and their ligands. These proteins mediate axonal guidance, synapse targeting, and other neuronal wiring-related functions. Recently, 32 interacting cell surface proteins belonging to two newly defined families of the Ig superfamily (IgSF) in fruit flies were discovered to label different subsets of neurons in the brain and ventral nerve cord. They have been shown to be involved in synaptic targeting and morphogenesis, retrograde signaling, and neuronal survival. Here, we show that these proteins, Dprs and DIPs, are members of a widely distributed family of two- and three-Ig domain molecules with neuronal wiring functions, which we refer to as Wirins. Beginning from a single ancestral Wirin gene in the last common ancestor of Bilateria, numerous gene duplications produced the heterophilic Dprs and DIPs in protostomes, along with two other subfamilies that diversified independently across protostome phyla. In deuterostomes, the ancestral Wirin evolved into the IgLON subfamily of neuronal receptors. We show that IgLONs interact with each other and that their complexes can be broken by mutations designed using homology models based on Dpr and DIP structures. The nematode orthologs ZIG-8 and RIG-5 also form heterophilic and homophilic complexes, and crystal structures reveal numerous apparently ancestral features shared with Dpr-DIP complexes. The evolutionary, biochemical, and structural relationships we demonstrate here provide insights into neural development and the rise of the metazoan nervous system.

Ancient mechanisms for the evolution of the bicoid homeodomain's function in fly development.

The ancient mechanisms that caused developmental gene regulatory networks to diversify among distantly related taxa are not well understood. Here we use ancestral protein reconstruction, biochemical experiments, and developmental assays of transgenic animals carrying reconstructed ancestral genes to investigate how the transcription factor Bicoid (Bcd) evolved its central role in anterior-posterior patterning in flies. We show that most of Bcd's derived functions are attributable to evolutionary changes within its homeodomain (HD) during a phylogenetic interval >140 million years ago. A single substitution from this period (Q50K) accounts almost entirely for the evolution of Bcd's derived DNA specificity in vitro. In transgenic embryos expressing the reconstructed ancestral HD, however, Q50K confers activation of only a few of Bcd's transcriptional targets and yields a very partial rescue of anterior development. Adding a second historical substitution (M54R) confers regulation of additional Bcd targets and further rescues anterior development. These results indicate that two epistatically interacting mutations played a major role in the evolution of Bcd's controlling regulatory role in early development. They also show how ancestral sequence reconstruction can be combined with in vivo characterization of transgenic animals to illuminate the historical mechanisms of developmental evolution.

Multinucleotide mutations cause false inferences of lineage-specific positive selection.

Phylogenetic tests of adaptive evolution, such as the widely used branch-site test (BST), assume that nucleotide substitutions occur singly and independently. Recent research has shown that errors at adjacent sites often occur during DNA replication, and the resulting multinucleotide mutations (MNMs) are overwhelmingly likely to be non-synonymous. To evaluate whether the BST misinterprets sequence patterns produced by MNMs as false support for positive selection, we analysed two genome-scale datasets-one from mammals and one from flies. We found that codons with multiple differences account for virtually all the support for lineage-specific positive selection in the BST. Simulations under conditions derived from these alignments but without positive selection show that realistic rates of MNMs cause a strong and systematic bias towards false inferences of selection. This bias is sufficient under empirically derived conditions to produce false positive inferences as often as the BST infers positive selection from the empirical data. Although some genes with BST-positive results may have evolved adaptively, the test cannot distinguish sequence patterns produced by authentic positive selection from those caused by neutral fixation of MNMs. Many published inferences of adaptive evolution using this technique may therefore be artefacts of model violation caused by unincorporated neutral mutational processes. We introduce a model that incorporates MNMs and may help to ameliorate this bias.

Pervasive contingency and entrenchment in a billion years of Hsp90 evolution.

Interactions among mutations within a protein have the potential to make molecular evolution contingent and irreversible, but the extent to which epistasis actually shaped historical evolutionary trajectories is unclear. To address this question, we experimentally measured how the fitness effects of historical sequence substitutions changed during the billion-year evolutionary history of the heat shock protein 90 (Hsp90) ATPase domain beginning from a deep eukaryotic ancestor to modern Saccharomyces cerevisiae We found a pervasive influence of epistasis. Of 98 derived amino acid states that evolved along this lineage, about half compromise fitness when introduced into the reconstructed ancestral Hsp90. And the vast majority of ancestral states reduce fitness when introduced into the extant S. cerevisiae Hsp90. Overall, more than 75% of historical substitutions were contingent on permissive substitutions that rendered the derived state nondeleterious, became entrenched by subsequent restrictive substitutions that made the ancestral state deleterious, or both. This epistasis was primarily caused by specific interactions among sites rather than a general effect on the protein's tolerance to mutation. Our results show that epistasis continually opened and closed windows of mutational opportunity over evolutionary timescales, producing histories and biological states that reflect the transient internal constraints imposed by the protein's fleeting sequence states.

Alternative evolutionary histories in the sequence space of an ancient protein.

To understand why molecular evolution turned out as it did, we must characterize not only the path that evolution followed across the space of possible molecular sequences but also the many alternative trajectories that could have been taken but were not. A large-scale comparison of real and possible histories would establish whether the outcome of evolution represents an optimal state driven by natural selection or the contingent product of historical chance events; it would also reveal how the underlying distribution of functions across sequence space shaped historical evolution. Here we combine ancestral protein reconstruction with deep mutational scanning to characterize alternative histories in the sequence space around an ancient transcription factor, which evolved a novel biological function through well-characterized mechanisms. We find hundreds of alternative protein sequences that use diverse biochemical mechanisms to perform the derived function at least as well as the historical outcome. These alternatives all require prior permissive substitutions that do not enhance the derived function, but not all require the same permissive changes that occurred during history. We find that if evolution had begun from a different starting point within the network of sequences encoding the ancestral function, outcomes with different genetic and biochemical forms would probably have resulted; this contingency arises from the distribution of functional variants in sequence space and epistasis between residues. Our results illuminate the topology of the vast space of possibilities from which history sampled one path, highlighting how the outcome of evolution depends on a serial chain of compounding chance events.