Computational Design of Protein Structure and Prediction of Ligand Binding

Proteins perform a tremendous array of finely-tuned functions which are not only critical in living organisms, but can be used for industrial and medical purposes. The ability to rationally design these molecular machines could provide a wealth of opportunities, for example to improve human health and to expand the range and reduce cost of many industrial chemical processes. The modularity of a protein sequence combined with many degrees of structural freedom yield a problem that can frequently be best tackled using computational methods. These computational methods, which include the use of: bioinformatics analysis, molecular dynamics, empirical forcefields, statistical potentials, and machine learning approaches, amongst others, are collectively known as Computational Protein Design (CPD). Here CPD is examined from the perspective of four different goals: successful design of an intended structure, the prediction of folding and unfolding kinetics from structure (kinetic stability in particular), engineering of improved stability, and prediction of binding sites and energetics. A considerable proportion of protein folds, and the majority of the most common folds ("superfolds"), are internally symmetric, suggesting emergence from an ancient repetition event. CPD, an increasingly popular and successful method for generating de novo folded sequences and topologies, suffers from exponential scaling of complexity with protein size. Thus, the overwhelming majority of successful designs are of relatively small proteins (< 100 amino acids). Designing proteins comprised of repeated modular elements allows the design space to be partitioned into more manageable portions. Here, a bioinformatics analysis of a "superfold", the beta-trefoil, demonstrated that formation of a globular fold via repetition was not only an ancient event, but an ongoing means of generating diverse and functional sequences. Modular repetition also promotes rapid evolution for binding multivalent targets in the "evolutionary arms race" between host and pathogen. Finally, modular repetition was used to successfully design, on the first attempt, a well-folded and functional beta-trefoil, called ThreeFoil. Improving protein design requires understanding the outcomes of design and not simply the 3D structure. To this end, I undertook an extensive biophysical characterization of ThreeFoil, with the key finding that its unfolding is extraordinarily slow, with a half-life of almost a decade. This kinetic stability grants ThreeFoil near-immunity to common denaturants as well as high resistance to proteolysis. A large scale analysis of hundreds of proteins, and coarse-grained modelling of ThreeFoil and other beta-trefoils, indicates that high kinetic stability results from a folded structure rich in contacts between residues distant in sequence (long-range contacts). Furthermore, an analysis of unrelated proteins known to have similar protease resistance, demonstrates that the topological complexity resulting from these long-range contacts may be a general mechanism by which proteins remain folded in harsh environments. Despite the wonderful kinetic stability of ThreeFoil, it has only moderate thermodynamic stability. I sought to improve this in order to provide a stability buffer for future functional engineering and mutagenesis. Numerous computational tools which predict stability change upon point mutation were used, and 10 mutations made based on their recommendations. Despite claims of >80% accuracy for these predictions, only 2 of the 10 mutations were stabilizing. An in-depth analysis of more than 20 such tools shows that, to a large extent, while they are capable of recognizing highly destabilizing mutations, they are unable to distinguish between moderately destabilizing and stabilizing mutations. Designing protein structure tests our understanding of the determinants of protein folding, but useful function is often the final goal of protein engineering. I explored protein-ligand binding using molecular dynamics for several protein-ligand systems involving both flexible ligand binding to deep pockets and more rigid ligand binding to shallow grooves. I also used various levels of simulation complexity, from gas-phase, to implicit solvent, to fully explicit solvent, as well as simple equilibrium simulations to interrogate known interactions to more complex energetically biased simulations to explore diverse configurations and gain novel information

From Peptides to Proteins: Exploring Modular Evolution Through the Beta-Trefoil Fold

Understanding the origin of protein folds, and the mechanism by which evolution has generated them, is a critically important step on a path towards rational protein design. Modifying existing proteins and designing our own novel folds and functions is a lofty but achievable goal, for which there are many foreseeable rewards. It is believed that modern proteins may have arisen from a primordial set of peptide precursors, which were initially only pseudo-stable or stable only as complexes with RNA, and later were able to self-assemble into multimeric complexes that resembled modern folds. In order to experimentally examine the feasibility of this theory, an attempt was made at reconstructing the evolutionary path of a beta-trefoil. The beta-trefoil is a naturally abundant fold or superfold, possessing pseudo-threefold symmetry, and usually having a sugar-binding function. It has been proposed that such a fold could arise from the triplication of just one small peptide on the order of 40-50 amino acids in length. The evolutionary path of a ricin, a family within the beta-trefoils known to possess a carbohydrate binding function was the chosen template for evolutionary modelling. It was desirable to have a known function associated with this design, such that it would be possible to determine if not only the fold, but also the function, could be reconstructed. A small peptide of 47 amino acids was designed and expressed. This peptide not only trimerized as expected, but possessed the carbohydrate binding function it was predicted to have. In an evolutionary model of the early protein world, the gene for this peptide would undergo duplication and later, triplication, eventually resulting in a completely symmetrical beta-trefoil, which would represent the first modern beta-trefoil fold. Such a completely symmetrical protein was also designed and expressed by triplicating the gene for the aforementioned small peptide. This hypothetical first modern beta-trefoil is: well folded, stable, soluble, and appears to adopt a beta-trefoil fold. Together these results demonstrate that an evolutionary model of early life: that proteins first existed as self-assembling modular peptides, and subsequent to gene duplications or fusions, as what we now recognize as modern folds, is experimentally consistent and not only generates stable structures, but those with function, which of course is a prime requisite of evolution. Moreover the results show that it may be possible to use this modular nature of protein folding to design our own proteins and predict the structure of others

Modular Evolution and the Origins of Symmetry: Reconstruction of a Three-Fold Symmetric Globular Protein

SummaryThe high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules

Biopsy confirmation of metastatic sites in breast cancer patients:clinical impact and future perspectives

Determination of hormone receptor (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2 status in the primary tumor is clinically relevant to define breast cancer subtypes, clinical outcome,and the choice of therapy. Retrospective and prospective studies suggest that there is substantial discordance in receptor status between primary and recurrent breast cancer. Despite this evidence and current recommendations,the acquisition of tissue from metastatic deposits is not routine practice. As a consequence, therapeutic decisions for treatment in the metastatic setting are based on the features of the primary tumor. Reasons for this attitude include the invasiveness of the procedure and the unreliable outcome of biopsy, in particular for biopsies of lesions at complex visceral sites. Improvements in interventional radiology techniques mean that most metastatic sites are now accessible by minimally invasive methods, including surgery. In our opinion, since biopsies are diagnostic and changes in biological features between the primary and secondary tumors can occur, the routine biopsy of metastatic disease needs to be performed. In this review, we discuss the rationale for biopsy of suspected breast cancer metastases, review issues and caveats surrounding discordance of biomarker status between primary and metastatic tumors, and provide insights for deciding when to perform biopsy of suspected metastases and which one (s) to biopsy. We also speculate on the future translational implications for biopsy of suspected metastatic lesions in the context of clinical trials and the establishment of bio-banks of biopsy material taken from metastatic sites. We believe that such bio-banks will be important for exploring mechanisms of metastasis. In the future,advances in targeted therapy will depend on the availability of metastatic tissue

An acute bout of swimming increases post-exercise energy intake in young healthy men and women

Single bouts of land-based exercise (for example, walking, running, cycling) do not typically alter post-exercise energy intake on the day of exercise. However, anecdotal and preliminary empirical evidence suggests that swimming may increase appetite and energy intake. This study compared the acute effects of swimming on appetite, energy intake, and food preference and reward, versus exertion-matched cycling and a resting control. Thirty-two men (n=17; mean ± SD age 24 ± 2 years, body mass index [BMI] 25.0 ± 2.6 kg/m2) and women (n=15; age 22 ± 3 years, BMI 22.8 ± 2.3 kg/m2) completed three experimental trials (swimming, cycling, control) in a randomised, crossover design. The exercise trials involved 60-min of ‘hard’ exercise (self-selected rating of perceived exertion: 15) performed 90-min after a standardised breakfast. Food preference and reward were assessed via the Leeds Food Preference Questionnaire 15-min after exercise, whilst ad libitum energy intake was determined 30-min after exercise. The control trial involved identical procedures except no exercise was performed. Compared with control (3259 ± 1265 kJ), swimming increased ad libitum energy intake (3857 ± 1611 kJ; ES=0.47, 95% CI of the mean difference between trials 185, 1010 kJ, P=0.005); the magnitude of increase was smaller after cycling (3652 ± 1619 kJ; ES=0.31, 95% CI -21, 805 kJ, P=0.062). Ad libitum energy intake was similar between swimming and cycling (ES=0.16, 95% CI -207, 618 kJ, P=0.324). This effect was consistent across sexes and unrelated to food preference and reward which were similar after swimming and cycling compared with control. This study has identified an orexigenic effect of swimming. Further research is needed to identify the responsible mechanism(s), including the relevance of water immersion and water temperature per se

Asymmetric Anchoring Is Required for Efficient Ω‑Loop Opening and Closing in Cytosolic Phosphoenolpyruvate Carboxykinase

Mobile Ω-loops play essential roles in the function of many enzymes. Here we investigated the importance of a residue lying outside of the mobile Ω-loop element in the catalytic function of an H477R variant of cytosolic phosphoenolpyruvate carboxykinase using crystallographic, kinetic, and computational analysis. The crystallographic data suggest that the efficient transition of the Ω-loop to the closed conformation requires stabilization of the N-terminus of the loop through contacts between R461 and E588. In contrast, the C-terminal end of the Ω-loop undergoes changing interactions with the enzyme body through contacts between H477 at the C-terminus of the loop and E591 located on the enzyme body. Potential of mean force calculations demonstrated that altering the anchoring of the C-terminus of the Ω-loop via the H477R substitution results in the destabilization of the closed state of the Ω-loop by 3.4 kcal mol^–1. The kinetic parameters for the enzyme were altered in an asymmetric fashion with the predominant effect being observed in the direction of oxaloacetate synthesis. This is exemplified by a reduction in <i>k</i>_cat for the H477R mutant by an order of magnitude in the direction of OAA synthesis, while in the direction of PEP synthesis, it decreased by a factor of only 2. The data are consistent with a mechanism for loop conformational exchange between open and closed states in which a balance between fixed anchoring of the N-terminus of the Ω-loop and a flexible, unattached C-terminus drives the transition between a disordered (open) state and an ordered (closed) state

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data