Computational design with flexible backbone sampling for protein remodeling and scaffolding of complex binding sites

Dissertation presented to obtain the Doutoramento (Ph.D.) degree in Biochemistry at the Instituto de Tecnologia Qu mica e Biol ogica da Universidade Nova de LisboaComputational protein design has achieved several milestones, including the design of a new protein fold, the design of enzymes for reactions that lack natural catalysts, and the re-engineering of protein-protein and protein-DNA binding speci city. These achievements have spurred demand to apply protein design methods to a wider array of research problems. However, the existing computational methods have largely relied on xed-backbone approaches that may limit the scope of problems that can be tackled. Here, we describe four computational protocols - side chain grafting, exible backbone remodeling, backbone grafting, and de novo sca old design - that expand the methodological protein design repertoire, three of which incorporate backbone exibility. Brie y, in the side chain grafting method, side chains of a structural motif are transplanted to a protein with a similar backbone conformation; in exible backbone remodeling, de novo segments of backbone are built and designed; in backbone grafting, structural motifs are explicitly grafted onto other proteins; and in de novo sca olding, a protein is folded and designed around a structural motif. We developed these new methods for the design of epitope-sca old vaccines in which viral neutralization epitopes of known three-dimensional structure were transplanted onto nonviral sca old proteins for conformational stabilization and immune presentation.(...

Design, ligand binding and folding of ankyrin and armadillo repeat proteins studied by solution NMR spectroscopy

Natural repeat proteins fulfil a plethora of important functions in cell biology like molecular recognition, cell adhesion and transport. Well known representatives of this protein class include armadillo, ankyrin, HEAT and tetratricopeptide repeat proteins. Proteins of this class constitute almost 20 % of proteins encoded in the human genome and contain tandem arrays of small, highly similar structural units. Several of these units stack against each other forming non-globular, elongated structures with long hydrophobic cores and extensive solvent exposed surfaces, determining topology and function of these proteins. Repeat proteins differ from globular proteins in several important characteristics. Firstly, they commonly display an extended solenoid fold. Secondly, they are mainly stabilized by short-range interactions between residues close in sequence, whereas the importance of long-range interactions for protein stability is greatly diminished compared to globular proteins. Two repeat protein families were investigated during this PhD project – ankyrin repeat protein and armadillo repeat proteins. The natural ankyrin repeat is a very common type of motif and can be found in all three kingdoms spanning a wide range of functions, with the underlying theme being their ability to mediate protein-protein interactions by binding to three-dimensional epitopes. Armadillo repeat proteins are commonly involved in protein-protein or protein-peptide interactions, binding to peptides or unfolded parts of proteins. Importantly, their extended binding surface can bind peptides in extended conformation. Protein engineering efforts aim at developing useful proteins with new or enhanced functions. The Plückthun group has undertaken an extensive design effort to create a highly stable designed consensus ankyrin repeat protein (DARPin) scaffold. These studies have cumulated in an optimized design, in which surface residues can be mutated to achieve binding to a desired target without compromising scaffold stability. Repeat proteins in general, and DARPins in particular, are an interesting subject upon which to study protein folding in order to understand the molecular base of their unusual stability. Their low contact order and modularity represents an intriguing background against which to study the mechanisms of protein folding and protein stability in a uniform environment. In this work, we investigated the stability and folding behaviour of full-consensus designed ankyrin repeat proteins (DARPins) using a range of NMR, biophysical and computational experiments. The sequence background of identical repeats used for our study can be seen as a generalised example for the study of AR protein folding and enables the investigation of folding as a function of repeat number. Using proton-exchange methods in the presence and absence of chemical denaturation, we evaluated the stability of this ankyrin scaffold in a residue-resolved manner. In order to achieve this we had to first assign the backbone resonances of each repeat and the N- and C-terminal capping repeats – a problem which becomes progressively more difficult as additional internal repeat are added. Paramagnetic spin labels attached to either end of the proteins were successfully used to decrease ambiguitiy and allowed complete backbone resonance assignments

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

Developing Computational Tools for the Study and Design of Amyloid Materials

The self-assembly of short peptides into amyloid structures is linked to several diseases but has also been exploited for the design of novel functional amyloid-based materials. Such materials are potentially biocompatible and biodegradable, while their unique molecular organization provides them with remarkable mechanical properties. Amyloid fibrils are among the stiffest biological materials and exhibit a high resistance to breakage. Apart from the aforementioned properties, they are particularly attractive due to their easy synthesis and the ability to be redesigned through mutations at sequence level, which can result in potential functionality. Previous studies have reported the rational based design of functional amyloid materials, designed through primarily scientists’ intuition, and their applications in several fields as agents for tissue-engineering, antimicrobial and antibacterial agents, drug carriers, materials for separation applications, etc. The current work starts from the use of previously reported protocols for the computational elucidation of the structure of amyloids, leading to the formation of amyloid materials, and the investigation of the functional properties of rationally designed self-assembling peptides, and introduces a new approach for the computational design of functional amyloid materials, based on engineering and biophysical principles. In summary, we developed a computational protocol according to which an optimization-based design model is used to introduce mutations at non-βsheet residue positions of an amyloid designable scaffold (amyloid with non-β-sheet forming residues at its termini). The designed amino acids are introduced to the scaffold in such a way so that they mimic how amino acids bind to particular ions/compounds of interest according to experimentally resolved structures (defined by us as materialphore models) and also aim at energetically stabilizing the bound conformation of the pockets. The optimum designs are computationally validated using a series of simulations and structural analysis techniques to select the top designed peptides, which are predicted to form fibrils with specific ion/compound binding properties for experimental testing. The computational protocol has been implemented first for the design of amyloid materials (i) binding to cesium ions, and in additional cases, for the design of amyloid materials (ii) serving as potential AD drug carriers, (iii) which could promote cell-penetration and possess DNA binding properties, and (iv) incorporating potential cell-adhesion, calcium and strontium binding properties. The computational protocol is also presented here as a step toward a generalized computational approach to design functional amyloid materials binding to an ion/compound of interest. This work can constitute a stepping stone for the functionalization of peptide/protein-based materials for several applications in the future