SummaryMolecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence.
The primary sequence of proteins usually dictates a single tertiary and quaternary structure. However, certain proteins undergo reversible backbone rearrangements. Such metamorphic proteins provide a means of facilitating the evolution of new folds and architectures. However, because natural folds emerged at the early stages of evolution, the potential role of metamorphic intermediates in mediating evolutionary transitions of structure remains largely unexplored. We evolved a set of new proteins based on ∼100 amino acid fragments derived from tachylectin-2-a monomeric, 236 amino acids, five-bladed β-propeller. Their structures reveal a unique pentameric assembly and novel β-propeller structures. Although identical in sequence, the oligomeric subunits adopt two, or even three, different structures that together enable the pentameric assembly of two propellers connected via a small linker. Most of the subunits adopt a wild-type-like structure within individual five-bladed propellers. However, the bridging subunits exhibit domain swaps and asymmetric strand exchanges that allow them to complete the two propellers and connect them. Thus, the modular and metamorphic nature of these subunits enabled dramatic changes in tertiary and quaternary structure, while maintaining the lectin function. These oligomers therefore comprise putative intermediates via which β-propellers can evolve from smaller elements. Our data also suggest that the ability of one sequence to equilibrate between different structures can be evolutionary optimized, thus facilitating the emergence of new structures.beta-propellers | conformational diversity | lectins | oligomerization | protein evolution M aynard-Smith's conjecture of protein evolution dictates that transitions of function and structure occur gradually (by single mutational steps), and smoothly, namely via intermediates that are all functional (1). This restriction imposes a major challenge, because mutational steps may create large structural perturbations that need to be tolerated while retaining function (2). In single domain proteins, significant "cut and paste" structural changes perturb a well-packed hydrophobic core (3, 4). Evolutionary transitions from one folded and functional domain structure into a new one should therefore (or must, by default, if one follows Maynard-Smith's conjecture) involve intermediates able to adopt more than one structure (5, 6). It has been also speculated that evolutionary intermediates of many existing folds, and folds that show internal symmetry in particular, were oligomeric assemblies of smaller subunits. Interestingly, most of the reported "metamorphic" proteins-proteins adopting more than one fold (7), are oliogomers in at least one of the two folds they adopt (8-10). Oligomerization may therefore serve as a means of augmenting structural metamorphism and of facilitating the emergence of new folds (11). However, metamorphic proteins are very rarely identified, and their evolutionary relevance is yet to be established. The involvement of struct...
Internal symmetry in proteins is likely to be the footprint of evolution by gene duplication and fusion. Like other symmetrical proteins, β-propellers, which are made of 4-10 β-sheet units (blades) circularly arranged around a central tunnel, have probably evolved by duplication and fusion of a rudimentary repetitive unit. However, reproducing the evolution of functional β-propellers by duplication and fusion of repeated units remains a challenge, in particular, because the repeated units must jointly pack to form one hydrophobic core while maintaining intact active sites. As model for generating repeat propellers, we chose tachylectin-2--a highly symmetrical five-bladed β-propeller lectin with five sugar-binding sites. We report the engineering of folded and functional lectins by duplication and fusion of repetitive sequence modules taken from tachylectin-2. The repeated modules comprise three strands of one blade plus one strand of the next blade, thus enabling the closure of the propeller's ring via strand-strand Velcro-like interactions. Duplication and fusion of five modules with the same sequence gave rise to a highly aggregated protein, yet its soluble fraction exhibited lectin function. Subsequently, a library of diversified sequence modules fused in tandem was selected by phage display for glycoprotein binding. A range of new lectins were isolated with binding and biophysical properties that resemble those of wild-type tachylectin-2. These results demonstrate the ability to construct folded and functional globular repeat proteins, and support the role of duplication and fusion of elementary modules in the evolutionary routes that led to the β-propellers fold.
Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN-and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s −1 ) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB.biodegradation | molecular evolution | channeling | quinone reductase
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