Valeria A. Risso

Hyperstability and Substrate Promiscuity in Laboratory Resurrections of Precambrian β‑Lactamases

We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A β-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (~100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2-3 billion years (Gyr)-old β-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (~35 °C) relative to modern β-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2-3 Gyr-old β-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution.

Evolution of Conformational Dynamics Determines the Conversion of a Promiscuous Generalist into a Specialist Enzyme

β-Lactamases are produced by many modern bacteria as a mechanism of resistance toward β-lactam antibiotics, the most common antibiotics in use. β-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A β-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern β-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant β-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient β-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity.

Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history

Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations.

Phenotypic comparisons of consensus variants versus laboratory resurrections of Precambrian proteins

Consensus‐sequence engineering has generated protein variants with enhanced stability, and sometimes, with modulated biological function. Consensus mutations are often interpreted as the introduction of ancestral amino acid residues. However, the precise relationship between consensus engineering and ancestral protein resurrection is not fully understood. Here, we report the properties of proteins encoded by consensus sequences derived from a multiple sequence alignment of extant, class A β‐lactamases, as compared with the properties of ancient Precambrian β‐lactamases resurrected in the laboratory. These comparisons considered primary sequence, secondary, and tertiary structure, as well as stability and catalysis against different antibiotics. Out of the three consensus variants generated, one could not be expressed and purified (likely due to misfolding and/or low stability) and only one displayed substantial stability having substrate promiscuity, although to a lower extent than ancient β‐lactamases. These results: (i) highlight the phenotypic differences between consensus variants and laboratory resurrections of ancestral proteins; (ii) question interpretations of consensus proteins as phenotypic proxies of ancestral proteins; and (iii) support the notion that ancient proteins provide a robust approach toward the preparation of protein variants having large numbers of mutational changes while possessing unique biomolecular properties. Proteins 2014; 82:887–896.

Selection for Protein Kinetic Stability Connects Denaturation Temperatures to Organismal Temperatures and Provides Clues to Archaean Life.

The relationship between the denaturation temperatures of proteins (Tm values) and the living temperatures of their host organisms (environmental temperatures: TENV values) is poorly understood. Since different proteins in the same organism may show widely different Tm’s, no simple universal relationship between Tm and TENV should hold, other than Tm≥TENV. Yet, when analyzing a set of homologous proteins from different hosts, Tm’s are oftentimes found to correlate with TENV’s but this correlation is shifted upward on the Tm axis. Supporting this trend, we recently reported Tm’s for resurrected Precambrian thioredoxins that mirror a proposed environmental cooling over long geological time, while remaining a shocking ~50°C above the proposed ancestral ocean temperatures. Here, we show that natural selection for protein kinetic stability (denaturation rate) can produce a Tm↔TENV correlation with a large upward shift in Tm. A model for protein stability evolution suggests a link between the Tm shift and the in vivo lifetime of a protein and, more specifically, allows us to estimate ancestral environmental temperatures from experimental denaturation rates for resurrected Precambrian thioredoxins. The TENV values thus obtained match the proposed ancestral ocean cooling, support comparatively high Archaean temperatures, and are consistent with a recent proposal for the environmental temperature (above 75°C) that hosted the last universal common ancestor. More generally, this work provides a framework for understanding how features of protein stability reflect the environmental temperatures of the host organisms.

De novo active sites for resurrected Precambrian enzymes

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Cooperativity and flexibility in enzyme evolution

Enzymes are flexible catalysts, and there has been substantial discussion about the extent to which this flexibility contributes to their catalytic efficiency. What has been significantly less discussed is the extent to which this flexibility contributes to their evolvability. Despite this, recent years have seen an increasing number of both experimental and computational studies that demonstrate that cooperativity and flexibility play significant roles in enzyme innovation. This review covers key developments in the field that emphasize the importance of enzyme dynamics not just to the evolution of new enzyme function(s), but also as a property that can be harnessed in the design of new artificial enzymes.

Biophysical characterization of the membrane-proximal ectodomain of the receptor-type protein-tyrosine phosphatase phogrin.

The receptor-type protein-tyrosine phosphatase (RPTP) phogrin is localized at the membrane of secretory granules of pancreatic islet β-cells and, similarly to the closely related ICA512, plays a role in the regulation of insulin secretion, in ensuring proper granulogenesis and stability, and in the regulation of β-cell growth. The mature membraneproximal ectodomain of phogrin (MPE phogrin) was produced as a recombinant protein and characterized. CD, fluorescence, controlled proteolysis, size-exclusion chromatography, and multi-angle light scattering showed that it is a properlyfolded monomeric domain. Equilibrium experiments, in the presence of guanidinium chloride and thermal unfolding, suggest a two-state mechanism with a ΔG of 2.3-3.3 kcal/mol, respectively. The study establishes common features and differences of MPE phogrin and the homologous ectodomain of ICA512. A homology model of phogrin was built based in the x-ray structure of MPE ICA512. The model is a starting point for modeling the entire receptor and for testing the quaternary structure and interactions of this protein in vivo. A description of the membrane insertion mode and putative interacting surfaces of this large protein is fundamental for the understanding of its biological function.

Preparation and biophysical characterization of recombinant Pseudomonas aeruginosa phosphorylcholine phosphatase

Pseudomonas aeruginosa infections constitute a widespread health problem with high economical and social impact, and the phosphorylcholine phosphatase (PchP) of this bacterium is a potential target for antimicrobial treatment. However, drug design requires high-resolution structural information and detailed biophysical knowledge not available for PchP. An obstacle in the study of PchP is that current methods for its expression and purification are suboptimal and allowed only a preliminary kinetic characterization of the enzyme. Herein, we describe a new procedure for the efficient preparation of recombinant PchP overexpressed in Escherichia coli. The enzyme is purified from urea solubilized inclusion bodies and refolded by dialysis. The product of PchP refolding is a mixture of native PchP and a kinetically-trapped, alternatively-folded aggregate that is very slowly converted into the native state. The properly folded and fully active enzyme is isolated from the refolding mixture by size-exclusion chromatography. PchP prepared by the new procedure was subjected to chemical and biophysical characterization, and its basic optical, hydrodynamic, metal-binding, and catalytic properties are reported. The unfolding of the enzyme was also investigated, and its thermal stability was determined. The obtained information should help to compare PchP with other phosphatases and to obtain a better understanding of its catalytic mechanism. In addition, preliminary trials showed that PchP prepared by the new protocol is suitable for crystallization, opening the way for high-resolution studies of the enzyme structure.

Optical studies of single-tryptophan B. licheniformis β-lactamase variants

beta-lactamases (penicillinases) are important complicating factors in bacterial infections and excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A beta-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp-->Phe mutants were prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolved fluorescence showed that most of the fluorescence decay of ESP tryptophans is due to a discrete exponential component with a lifetime of 5-6ns. Fluorescence polarization measurements indicated that fluorescence of Trp 210 is nearly independent of the fluorescence of Trp 229 and Trp 251, whereas a substantial energy homotransfer between the latter pair takes place. The spectroscopic information was rationalized on the basis of structural considerations and should help in the interpretation and monitoring of the changes at the sub domain level during the conformational transitions and fluctuations of ESP and other Class A beta-lactamases.