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1.
Molecular Determinants for Ligand Selectivity of the Cell-Free Synthesized Human Endothelin B Receptor.
Dong, F, Rues, RB, Kazemi, S, Dötsch, V, Bernhard, F
Journal of molecular biology. 2018;(24):5105-5119
Abstract
Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms.
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2.
Population-specific design of de-immunized protein biotherapeutics.
Schubert, B, Schärfe, C, Dönnes, P, Hopf, T, Marks, D, Kohlbacher, O
PLoS computational biology. 2018;(3):e1005983
Abstract
Immunogenicity is a major problem during the development of biotherapeutics since it can lead to rapid clearance of the drug and adverse reactions. The challenge for biotherapeutic design is therefore to identify mutants of the protein sequence that minimize immunogenicity in a target population whilst retaining pharmaceutical activity and protein function. Current approaches are moderately successful in designing sequences with reduced immunogenicity, but do not account for the varying frequencies of different human leucocyte antigen alleles in a specific population and in addition, since many designs are non-functional, require costly experimental post-screening. Here, we report a new method for de-immunization design using multi-objective combinatorial optimization. The method simultaneously optimizes the likelihood of a functional protein sequence at the same time as minimizing its immunogenicity tailored to a target population. We bypass the need for three-dimensional protein structure or molecular simulations to identify functional designs by automatically generating sequences using probabilistic models that have been used previously for mutation effect prediction and structure prediction. As proof-of-principle we designed sequences of the C2 domain of Factor VIII and tested them experimentally, resulting in a good correlation with the predicted immunogenicity of our model.
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3.
Rosetta FunFolDes - A general framework for the computational design of functional proteins.
Bonet, J, Wehrle, S, Schriever, K, Yang, C, Billet, A, Sesterhenn, F, Scheck, A, Sverrisson, F, Veselkova, B, Vollers, S, et al
PLoS computational biology. 2018;(11):e1006623
Abstract
The robust computational design of functional proteins has the potential to deeply impact translational research and broaden our understanding of the determinants of protein function and stability. The low success rates of computational design protocols and the extensive in vitro optimization often required, highlight the challenge of designing proteins that perform essential biochemical functions, such as binding or catalysis. One of the most simplistic approaches for the design of function is to adopt functional motifs in naturally occurring proteins and transplant them to computationally designed proteins. The structural complexity of the functional motif largely determines how readily one can find host protein structures that are "designable", meaning that are likely to present the functional motif in the desired conformation. One promising route to enhance the "designability" of protein structures is to allow backbone flexibility. Here, we present a computational approach that couples conformational folding with sequence design to embed functional motifs into heterologous proteins-Rosetta Functional Folding and Design (FunFolDes). We performed extensive computational benchmarks, where we observed that the enforcement of functional requirements resulted in designs distant from the global energetic minimum of the protein. An observation consistent with several experimental studies that have revealed function-stability tradeoffs. To test the design capabilities of FunFolDes we transplanted two viral epitopes into distant structural templates including one de novo "functionless" fold, which represent two typical challenges where the designability problem arises. The designed proteins were experimentally characterized showing high binding affinities to monoclonal antibodies, making them valuable candidates for vaccine design endeavors. Overall, we present an accessible strategy to repurpose old protein folds for new functions. This may lead to important improvements on the computational design of proteins, with structurally complex functional sites, that can perform elaborate biochemical functions related to binding and catalysis.
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4.
Engineered control of enzyme structural dynamics and function.
Boehr, DD, D'Amico, RN, O'Rourke, KF
Protein science : a publication of the Protein Society. 2018;(4):825-838
Abstract
Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine.
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5.
Design of catalytic polypeptides and proteins.
Gutte, B, Klauser, S
Protein engineering, design & selection : PEDS. 2018;(12):457-470
Abstract
The first part of this review article lists examples of complete, empirical de novo design that made important contributions to the development of the field and initiated challenging projects. The second part of this article deals with computational design of novel enzymes in native protein scaffolds; active designs were refined through random and site-directed mutagenesis producing artificial enzymes with nearly native enzyme- like activities against a number of non-natural substrates. Combining aspects of de novo design and biological evolution of nature's enzymes has started and will accelerate the development of novel enzyme activities.
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6.
Elfin: An algorithm for the computational design of custom three-dimensional structures from modular repeat protein building blocks.
Yeh, CT, Brunette, TJ, Baker, D, McIntosh-Smith, S, Parmeggiani, F
Journal of structural biology. 2018;(2):100-107
Abstract
Computational protein design methods have enabled the design of novel protein structures, but they are often still limited to small proteins and symmetric systems. To expand the size of designable proteins while controlling the overall structure, we developed Elfin, a genetic algorithm for the design of novel proteins with custom shapes using structural building blocks derived from experimentally verified repeat proteins. By combining building blocks with compatible interfaces, it is possible to rapidly build non-symmetric large structures (>1000 amino acids) that match three-dimensional geometric descriptions provided by the user. A run time of about 20min on a laptop computer for a 3000 amino acid structure makes Elfin accessible to users with limited computational resources. Protein structures with controlled geometry will allow the systematic study of the effect of spatial arrangement of enzymes and signaling molecules, and provide new scaffolds for functional nanomaterials.
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7.
Engineering altered protein-DNA recognition specificity.
Bogdanove, AJ, Bohm, A, Miller, JC, Morgan, RD, Stoddard, BL
Nucleic acids research. 2018;(10):4845-4871
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Abstract
Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein-protein interactions, modifying protein-DNA interactions is more difficult. This may be related to the structural features of protein-DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein-DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.
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8.
Commodity Chemicals From Engineered Modular Type I Polyketide Synthases.
Yuzawa, S, Zargar, A, Pang, B, Katz, L, Keasling, JD
Methods in enzymology. 2018;:393-415
Abstract
Reduced polyketides are a subclass of natural products that have a variety of medical, veterinary, and agricultural applications and are well known for their structural diversity. Although these compounds do not resemble each other, they are all made by a class of enzymes known as modular polyketide synthases (PKSs). The commonality of PKS domains/modules that compose PKSs and the understanding of the relationship between the sequence of the PKS and the structure of the compound it produces render modular PKSs as excellent targets for engineering to produce novel compounds with predicted structures. Here, we describe experimental protocols and considerations for modular PKS engineering and two case studies to produce commodity chemicals by engineered PKSs.
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9.
Protein engineering of CYP105s for their industrial uses.
Yasuda, K, Sugimoto, H, Hayashi, K, Takita, T, Yasukawa, K, Ohta, M, Kamakura, M, Ikushiro, S, Shiro, Y, Sakaki, T
Biochimica et biophysica acta. Proteins and proteomics. 2018;(1):23-31
Abstract
Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
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10.
Are natural proteins special? Can we do that?
Hecht, MH, Zarzhitsky, S, Karas, C, Chari, S
Current opinion in structural biology. 2018;:124-132
Abstract
Natural proteins represent a minuscule fraction of possible sequence space. These very rare sequences display remarkable properties: They fold into many different stable structures, and perform a wide range of complex biological functions. These two considerations-rarity and functionality-may suggest that natural proteins are somehow special. Is this true? We address this question by exploring attempts to recapitulate the special structures and functions of natural proteins into sequences designed de novo.