Emergence of fractal geometries in the evolution of a metabolic enzyme.

Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2-4; however, so far, molecular assembly into fractals is restricted to synthetic systems5-12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpinski triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.

Evolution-inspired engineering of nonribosomal peptide synthetases.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

The virulence regulator VirB from Shigella flexneri uses a CTP-dependent switch mechanism to activate gene expression.

The transcriptional antisilencer VirB acts as a master regulator of virulence gene expression in the human pathogen Shigella flexneri. It binds DNA sequences (virS) upstream of VirB-dependent promoters and counteracts their silencing by the nucleoid-organizing protein H-NS. However, its precise mode of action remains unclear. Notably, VirB is not a classical transcription factor but related to ParB-type DNA-partitioning proteins, which have recently been recognized as DNA-sliding clamps using CTP binding and hydrolysis to control their DNA entry gate. Here, we show that VirB binds CTP, embraces DNA in a clamp-like fashion upon its CTP-dependent loading at virS sites and slides laterally on DNA after clamp closure. Mutations that prevent CTP-binding block VirB loading in vitro and abolish the formation of VirB nucleoprotein complexes as well as virulence gene expression in vivo. Thus, VirB represents a CTP-dependent molecular switch that uses a loading-and-sliding mechanism to control transcription during bacterial pathogenesis.

Ion binding with charge inversion combined with screening modulates DEAD box helicase phase transitions.

Membraneless organelles, or biomolecular condensates, enable cells to compartmentalize material and processes into unique biochemical environments. While specific, attractive molecular interactions are known to stabilize biomolecular condensates, repulsive interactions, and the balance between these opposing forces, are largely unexplored. Here, we demonstrate that repulsive and attractive electrostatic interactions regulate condensate stability, internal mobility, interfaces, and selective partitioning of molecules both in vitro and in cells. We find that signaling ions, such as calcium, alter repulsions between model Ddx3 and Ddx4 condensate proteins by directly binding to negatively charged amino acid sidechains and effectively inverting their charge, in a manner fundamentally dissimilar to electrostatic screening. Using a polymerization model combined with generalized stickers and spacers, we accurately quantify and predict condensate stability over a wide range of pH, salt concentrations, and amino acid sequences. Our model provides a general quantitative treatment for understanding how charge and ions reversibly control condensate stability.

Molecular basis and design principles of switchable front-rear polarity and directional migration in Myxococcus xanthus.

During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions varies. In rod-shaped Myxococcus xanthus cells, a spatial toggle switch dynamically regulates front-rear polarity. The polarity module establishes front-rear polarity by guaranteeing front pole-localization of the small GTPase MglA. Conversely, the Frz chemosensory system, by acting on the polarity module, causes polarity inversions. MglA localization depends on the RomR/RomX GEF and MglB/RomY GAP complexes that localize asymmetrically to the poles by unknown mechanisms. Here, we show that RomR and the MglB and MglC roadblock domain proteins generate a positive feedback by forming a RomR/MglC/MglB complex, thereby establishing the rear pole with high GAP activity that is non-permissive to MglA. MglA at the front engages in negative feedback that breaks the RomR/MglC/MglB positive feedback allosterically, thus ensuring low GAP activity at this pole. These findings unravel the design principles of a system for switchable front-rear polarity.

Functional Degeneracy in Paracoccus denitrificans Pd1222 Is Coordinated via RamB, Which Links Expression of the Glyoxylate Cycle to Activity of the Ethylmalonyl-CoA Pathway.

Metabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.

Fortuitously compatible protein surfaces primed allosteric control in cyanobacterial photoprotection.

Highly specific interactions between proteins are a fundamental prerequisite for life, but how they evolve remains an unsolved problem. In particular, interactions between initially unrelated proteins require that they evolve matching surfaces. It is unclear whether such surface compatibilities can only be built by selection in small incremental steps, or whether they can also emerge fortuitously. Here, we used molecular phylogenetics, ancestral sequence reconstruction and biophysical characterization of resurrected proteins to retrace the evolution of an allosteric interaction between two proteins that act in the cyanobacterial photoprotection system. We show that this interaction between the orange carotenoid protein (OCP) and its unrelated regulator, the fluorescence recovery protein (FRP), evolved when a precursor of FRP was horizontally acquired by cyanobacteria. FRP's precursors could already interact with and regulate OCP even before these proteins first encountered each other in an ancestral cyanobacterium. The OCP-FRP interaction exploits an ancient dimer interface in OCP, which also predates the recruitment of FRP into the photoprotection system. Together, our work shows how evolution can fashion complex regulatory systems easily out of pre-existing components.

Evidence for an early green/red photocycle that precedes the diversification of GAF domain photoreceptor cyanobacteriochromes.

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation.

ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks.

RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo, RecA forms filamentous structures termed "threads," which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro, features lower dsDNA binding activity, but still retains most of wild type RecA activity in vivo. Contrarily, loss of ATPase activity strongly reduced formation of nucleoprotein filaments in vitro, and effectivity to repair double strand breaks (DSBs) in vivo. In the presence of wild type RecA protein, additionally expressed RecA with lowered ATPbinding activity only moderately affected RecA dynamics, while loss of ATPase activity leads to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and nonspecifically DNA-bound molecules into threads upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Based on the idea that ATPase activity of RecA is most important for DNA strand exchange activity, our data suggest that extension and retraction of threads due is to many local strand invasion events during the search for sequences homologous to the induced DNA break site. IMPORTANCE Single-strand (ss) DNA binding ATPase RecA is the central recombinase in homologous recombination, and therefore essential for DNA repair pathways involving DNA strand exchange reactions. In several bacterial, RecA forms filamentous structures along the long axis of cells after induction of double strand breaks (DSBs) in the chromosome. These striking assemblies likely reflect RecA/ssDNA nucleoprotein filaments, which can extend and remodel within a time frame of few minutes. We show that ATPase activity of RecA is pivotal for these dynamic rearrangements, which include recruitment of freely diffusing molecules into low-mobile molecules within filaments. Our data suggest that ssDNA binding- and unbinding reactions are at the heart of RecA dynamics that power the dynamics of subcellular filamentous assemblies, leading to strand exchange reactions over a distance of several micrometers.

Structural basis for lipid and copper regulation of the ABC transporter MsbA.

A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although LPS binding to the inner cavity of MsbA is well established, the selectivity of MsbA-lipid interactions at other site(s) remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5'-diphosphate and vanadate, revealing a distinct KDL binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KDL binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis.

Evolution of increased complexity and specificity at the dawn of form I Rubiscos.

The evolution of ribulose-1,5-bisphosphate carboxylase/oxygenases (Rubiscos) that discriminate strongly between their substrate carbon dioxide and the undesired side substrate dioxygen was an important event for photosynthetic organisms adapting to an oxygenated environment. We use ancestral sequence reconstruction to recapitulate this event. We show that Rubisco increased its specificity and carboxylation efficiency through the gain of an accessory subunit before atmospheric oxygen was present. Using structural and biochemical approaches, we retrace how this subunit was gained and became essential. Our work illuminates the emergence of an adaptation to rising ambient oxygen levels, provides a template for investigating the function of interactions that have remained elusive because of their essentiality, and sheds light on the determinants of specificity in Rubisco.

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.

Non-adaptive complexity and biochemical function.

Intricate biochemical structures are usually thought to be useful, because natural selection preserves them from degradation by a constant hail of destructive mutations. Biochemists therefore often deliberately disrupt them to understand how complexity improves protein function or fitness. However, evolutionary theory suggests that even useless complexity that never improved fitness can become completely essential if a simple set of evolutionary conditions is fulfilled. We review evidence that stable protein complexes, protein-chaperone interactions, and complexes consisting of several paralogs all fulfill these conditions. This makes reverse genetics or destructive mutagenesis unsuitable for assigning functions to these kinds of complexity. Instead, we advocate that incorporating evolutionary approaches into biochemistry overcomes this difficulty and allows us to distinguish useless from useful biochemical complexity.

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.

An ultra-stable gold-coordinated protein cage displaying reversible assembly.

Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery1, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging2,3. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Aui-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.

It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate.

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones, and sHsp mutations or altered expression are linked to multiple human disease states. sHsp monomers assemble into large oligomers with dimeric substructure, and the dynamics of sHsp oligomers has led to major questions about the form that captures substrate, a critical aspect of their mechanism of action. We show here that substructural dimers of two plant dodecameric sHsps, Ta16.9 and homologous Ps18.1, are functional units in the initial encounter with unfolding substrate. We introduced inter-polypeptide disulfide bonds at the two dodecameric interfaces, dimeric and nondimeric, to restrict how their assemblies can dissociate. When disulfide-bonded at the nondimeric interface, mutants of Ta16.9 and Ps18.1 (TaCT-ACD and PsCT-ACD) were inactive but, when reduced, had WT-like chaperone activity, demonstrating that dissociation at nondimeric interfaces is essential for sHsp activity. Moreover, the size of the TaCT-ACD and PsCT-ACD covalent unit defined a new tetrahedral geometry for these sHsps, different from that observed in the Ta16.9 X-ray structure. Importantly, oxidized Tadimer (disulfide bonded at the dimeric interface) exhibited greatly enhanced ability to protect substrate, indicating that strengthening the dimeric interface increases chaperone efficiency. Temperature-induced size and secondary structure changes revealed that folded sHsp dimers interact with substrate and that dimer stability affects chaperone efficiency. These results yield a model in which sHsp dimers capture substrate before assembly into larger, heterogeneous sHsp-substrate complexes for substrate refolding or degradation, and suggest that tuning the strength of the dimer interface can be used to engineer sHsp chaperone efficiency.

Structural principles that enable oligomeric small heat-shock protein paralogs to evolve distinct functions.

Oligomeric proteins assemble with exceptional selectivity, even in the presence of closely related proteins, to perform their cellular roles. We show that most proteins related by gene duplication of an oligomeric ancestor have evolved to avoid hetero-oligomerization and that this correlates with their acquisition of distinct functions. We report how coassembly is avoided by two oligomeric small heat-shock protein paralogs. A hierarchy of assembly, involving intermediates that are populated only fleetingly at equilibrium, ensures selective oligomerization. Conformational flexibility at noninterfacial regions in the monomers prevents coassembly, allowing interfaces to remain largely conserved. Homomeric oligomers must overcome the entropic benefit of coassembly and, accordingly, homomeric paralogs comprise fewer subunits than homomers that have no paralogs.