Protein design methods and applications

Methods in Molecular Biology 1216 Valentin Köhler Editor Protein Design Methods and Applications Second Edition Tai Lieu Chat Luong METHODS IN M O L E C U L A R B I O LO G Y Series Editor John M Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Protein Design Methods and Applications Second Edition Edited by Valentin Köhler Department of Chemistry, University of Basel, Switzerland Editor Valentin Köhler Department of Chemistry University of Basel Switzerland ISSN 1064-3745 ISSN 1940-6029 (electronic) ISBN 978-1-4939-1485-2 ISBN 978-1-4939-1486-9 (eBook) DOI 10.1007/978-1-4939-1486-9 Springer New York Heidelberg Dordrecht London Library of Congress Control Number: 2014947803 © Springer Science+Business Media New York 2014 This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made The publisher makes no warranty, express or implied, with respect to the material contained herein Printed on acid-free paper Humana Press is a brand of Springer Springer is part of Springer Science+Business Media (www.springer.com) Preface The second edition of protein design in the Methods in Molecular Biology series aims at providing the reader with practical guidance and general ideas on how to approach a potential protein design project Considering the complexity of the subject and its attention in the scientific community it is apparent that only a selection of subjects, approaches, methods, studies, and ideas can be presented The design of well-folded peptide structures and the redesign of existing proteins serve multiple purposes from potentially unlimited and only just developing applications in medicine, material science, catalysis, the realization of systems chemistry, and synthetic biology to a deeper understanding of molecular evolution The book is roughly organized in increasing complexity of the systems studied Additional emphasis is put on metals as structure-forming elements and functional sites of proteins towards the end A computational algorithm for the design of stable alpha helices is discussed in the first chapter and is accessible in the form of a web-based tool An extensive review on monomeric β-hairpin and β-sheet peptides follows In the design of these species any tendency to self-assemble has to be carefully considered In contrast, Chapter exploits just this phenomenon—peptides engineered to self-assemble into fibrils Subsequently, some possibilities and aspects resulting from the incorporation of unnatural amino acids are outlined In the practical methods chapter on the redesign of RNase A, a variable α-helical fragment is reassembled with the remainder of the protein structure, generated by enzymatic cleavage Chapter discusses the design and characterization of fluorinated proteins, which are entirely synthetic Comparisons to non-fluorinated analogous structures are included and practical advice is offered This is followed by an overview of considerations for the generation of binary-patterned protein libraries leading on to library-scale computational protein design for the engineering of improved protein variants The latter is exemplified for cellobiohydrolase II and a study aimed at changing the co-substrate specificity of a ketol-acid reductoisomerase Chapter focuses on the elaboration of symmetric protein folds in an approach termed “top-down symmetric deconstruction,” which prepares the folds for subsequent functional design studies The identification of a suitable scaffold for design purposes by means of the scaffold search program ScaffoldSelection is the topic of Chapter The computational design of novel enzymes without cofactor is demonstrated for a Diels-Alderase in Chapter 10 The final four chapters deal with metal involvement in the designed or redesigned structures, either as structural elements or functional centers The begin is made with a tutorial review that imparts general knowledge for the design of peptide scaffolds as novel pre-organized ligands for metal-ion coordination and then exemplifies these further in a respective case study This is followed by an introduction on the computational design of metalloproteins, which encompasses metal incorporation into existing folds, fold design by v vi Preface exploiting symmetry, and fold design in asymmetric scaffolds The potential power of cofactor exchange is addressed with the focus on a practical protocol for the preparation of apomyoglobin and the incorporation of zinc porphyrin in the penultimate chapter The book concludes with a case study on the computational redesign of metalloenzymes carried out with the aim to assign a new enzymatic function This volume of Methods in Molecular Biology contains a number of practical protocols, but compared to other volumes of the series, a larger contribution of reviews or general introductions is provided Those, however, are presented in a tutorial fashion to communicate principles that can be applied to individual research projects I sincerely hope that the reader finds this edition of protein design helpful for devising their own experiments I warmly thank all the authors for their very valuable contributions, their dedication, and not least their patience Basel, Switzerland Valentin Köhler Contents Preface Contributors v ix De Novo Design of Stable α-Helices Alexander Yakimov, Georgy Rychkov, and Michael Petukhov Design of Monomeric Water-Soluble β-Hairpin and β-Sheet Peptides M Angeles Jiménez Combination of Theoretical and Experimental Approaches for the Design and Study of Fibril-Forming Peptides Phanourios Tamamis, Emmanouil Kasotakis, Georgios Archontis, and Anna Mitraki Posttranslational Incorporation of Noncanonical Amino Acids in the RNase S System by Semisynthetic Protein Assembly Maika Genz and Norbert Sträter Design, Synthesis, and Study of Fluorinated Proteins Benjamin C Buer and E Neil G Marsh High-Quality Combinatorial Protein Libraries Using the Binary Patterning Approach Luke H Bradley Methods for Library-Scale Computational Protein Design Lucas B Johnson, Thaddaus R Huber, and Christopher D Snow Symmetric Protein Architecture in Protein Design: Top-Down Symmetric Deconstruction Liam M Longo and Michael Blaber Identification of Protein Scaffolds for Enzyme Design Using Scaffold Selection André C Stiel, Kaspar Feldmeier, and Birte Höcker 10 Computational Design of Novel Enzymes Without Cofactors Matthew D Smith, Alexandre Zanghellini, and Daniela Grabs-Röthlisberger 11 De Novo Design of Peptide Scaffolds as Novel Preorganized Ligands for Metal-Ion Coordination Aimee J Gamble and Anna F.A Peacock 12 Computational Design of Metalloproteins Avanish S Parmar, Douglas Pike, and Vikas Nanda vii 15 53 71 89 117 129 161 183 197 211 233 viii Contents 13 Incorporation of Modified and Artificial Cofactors into Naturally Occurring Protein Scaffolds Koji Oohora and Takashi Hayashi 14 Computational Redesign of Metalloenzymes for Catalyzing New Reactions Per Jr Greisen and Sagar D Khare Index 251 265 275 Contributors GEORGIOS ARCHONTIS • Department of Physics, University of Cyprus, Nicosia, Cyprus MICHAEL BLABER • Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA LUKE H BRADLEY • Departments of Anatomy and Neurobiology, Molecular and Cellular Biochemistry, and the Center of Structural Biology, University of Kentucky College of Medicine, Lexington, KY, USA BENJAMIN C BUER • Department of Chemistry, University of Michigan, Ann Arbor, MI, USA KASPAR FELDMEIER • Max Planck Institute for Developmental Biology, Tübingen, Germany AIMEE J GAMBLE • School of Chemistry, University of Birmingham, Birmingham, UK MAIKA GENZ • Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Institute of Bioanalytical Chemistry, University of Leipzig, Leipzig, Germany DANIELA GRABS-RƯTHLISBERGER • Arzeda Corp., Seattle, WA, USA PER JR GREISEN • Department of Biochemistry, University of Washington, Seattle, WA, USA TAKASHI HAYASHI • Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan BIRTE HƯCKER • Max Planck Institute for Developmental Biology, Tübingen, Germany THADDAUS R HUBER • Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO, USA M ANGELES JIMÉNEZ • Consejo Superior de Investigaciones Científicas (CSIC), Instituto de Química Física Rocasolano (IQFR), Madrid, Spain LUCAS B JOHNSON • Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO, USA EMMANOUIL KASOTAKIS • Department of Materials Science and Technology, University of Crete, Heraklion, Crete, Greece SAGAR D KHARE • Department of Chemistry and Chemical Biology, Center for Integrative Proteomics Research, Rutgers University, Piscataway, NJ, USA LIAM M LONGO • Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL, USA E NEIL G MARSH • Department of Chemistry, University of Michigan, Ann Arbor, MI, USA; Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA ANNA MITRAKI • Department of Materials Science and Technology, University of Crete, Heraklion, Crete, Greece; Institute for Electronic Structure and Laser, Foundation for Research and Technology-Hellas (IESL-FORTH), Heraklion, Crete, Greece VIKAS NANDA • Department of Biochemistry and Molecular Biology, Center for Advanced Biotechnology and Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA KOJI OOHORA • Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan ix Modified and Artificial Cofactors 17 18 19 20 myoglobin mutant H64D reconstituted with a “single-winged cofactor” is equivalent to native horseradish peroxidase in oxidation activity Chem Asian J 6:2491–2499 Hayashi T, Dejima H, Matsuo T, Sato H, Murata D, Hisaeda Y (2002) Blue myoglobin reconstituted with an iron porphycene shows extremely high oxygen affinity J Am Chem Soc 124:11226–11227 Ueno T, Koshiyama T, Ohashi M, Kondo K, Kono M, Suzuki A et al (2005) Coordinated design of cofactor and active site structures in development of new protein catalysts J Am Chem Soc 127:6556–6562 Patolsky F, Weizmann Y, Willner I (2004) Long-range electrical contacting of redox enzymes by SWCNT connectors Angew Chem Int Ed 43:2113–2117 Onoda A, Kakikura Y, Uematsu T, Kuwabata S, Hayashi T (2012) Photocurrent generation from hierarchical zinc-substituted hemoprotein assemblies immobilized on a gold 21 22 23 24 25 263 electrode Angew Chem Int Ed 51: 2628–2631 Kitagishi H, Oohora K, Yamaguchi H, Sato H, Matsuo T, Harada A et al (2007) Supramolecular hemoprotein linear assembly by successive interprotein heme–heme pocket interactions J Am Chem Soc 129:10326–10327 Oohora K, Onoda A, Hayashi T (2012) Supramolecular assembling systems formed by heme–heme pocket interactions in hemoproteins Chem Commun 48:11714–11726 Shibata T, Matsumoto D, Nishimura R, Tai H, Matsuoka A, Nagao S et al (2012) Relationship between oxygen affinity and autoxidation of myoglobin Inorg Chem 51:11955–11960 Smith KM (1975) Porphyrins and metalloporphyrins Elsevier, Amsterdam Fändrich M, Forge V, Buder K, Kittler M, Dobson CM, Diekmann S (2003) Myoglobin forms amyloid fibrils by association of unfolded polypeptide segments Proc Natl Acad Sci U S A 100:15463–15468 Chapter 14 Computational Redesign of Metalloenzymes for Catalyzing New Reactions Per Jr Greisen and Sagar D Khare Abstract The ability to design novel activities in existing metalloenzyme active sites is a stringent test of our understanding of enzyme mechanisms, sheds light on enzyme evolution, and would have many practical applications Here, we describe a computational method in the context of the macromolecular modeling suite Rosetta to repurpose active sites containing metal ions for reactions of choice The required inputs for the method are a model of the transition state(s) for the reaction and a set of crystallographic structures of proteins containing metal ions The coordination geometry associated with the metal ion (Zn2+, for example) is automatically detected and the transition state model is aligned to the open metal coordination site(s) in the protein Additional interactions to the transition state model are made using RosettaMatch and the surrounding amino acid side chain identities are optimized for transition state stabilization using RosettaDesign Validation of the design is performed using docking and molecular dynamics simulations, and candidate designs are generated for experimental validation Computational metalloenzyme repurposing is complementary to directed evolution approaches for enzyme engineering and allows large jumps in sequence space to make concerted sequence and structural changes for introducing novel enzymatic activities and specificities Key words Enzyme design, Rosetta software, Enzyme redesign, Metalloenzymes, Zinc ions Introduction Metal ions are versatile catalysts for carrying out biological and non-biological reactions, affording rates and reaction mechanisms not accessible in conventional acid–base or covalent catalysis Considerable effort has been made to design artificial metalloproteins [1, 2], however the de novo design of metal-dependent enzyme active sites has been challenging because of stringent design requirements: (a) multiple flexible, polar residues are necessary to bind the metal and these must be held in place by additional second-shell residues, (b) destabilization of alternative conformations that would disrupt the designed conformation is necessary (negative design), and (c) second- and third-shell effects can be critical for modulating the electrostatic environment of the active Valentin Köhler (ed.), Protein Design: Methods and Applications, Methods in Molecular Biology, vol 1216, DOI 10.1007/978-1-4939-1486-9_14, © Springer Science+Business Media New York 2014 265 266 Per Jr Greisen and Sagar D Khare site that is a key determinant of metal reactivity In view of these requirements, the repurposing of existing metal binding sites for accessing new chemical reactivity offers a relatively simple, yet effective strategy for design One of the most common metal ions in biology is the zinc ion (Zn2+) Zn2+ is coordinated by proteins in three different coordination geometries, tetrahedral, trigonal bipyramidal, and octahedral The reactivity of the site is regulated by the coordinating groups of the metal which are usually histidine (His), aspartic acid (Asp), glutamic acid (Glu), or cysteine (Cys) [3] The coordination sphere of the metal ion is flexible and it is possible to tune the reactivity of the site by means of the ligands In enzymes, Zn2+ can act as a Lewis acid for example in alcohol dehydrogenase, or it activates a water molecule to perform nucleophilic attack on a substrate for example in carbonic anhydrase, or both [4, 5] Therefore, zinc metalloenzymes provide a viable platform for the introduction of novel activities using computational repurposing Reusing some or all of the catalytic elements in existing enzyme active sites for new chemistry is a common theme in natural enzyme evolution, and underlies the functional diversification seen in enzyme superfamilies In contrast, de novo computational enzyme design aims at placing catalytic elements in otherwise inert scaffolds to introduce new reactivity We have implemented a computational design strategy that is inspired by natural enzyme evolution in that it reuses existing catalytic elements of enzyme active sites but also uses de novo enzyme design methods to rationally engineer new activities in the framework of the macromolecular modeling suite Rosetta Application of this method to a set of mononuclear zinc enzymes led the design of organophosphate hydrolysis activity in an adenosine deaminase [6] (Figs and 2) The method described below illustrates the approach used for the above design project, but has been extended to binuclear metal sites and for a variety of other reactions including s-triazine, beta-lactam, and cyanuric acid hydrolysis (unpublished data) Methods Starting from a transition state model of the reaction under consideration and a set of zinc-containing PDB files as inputs, we generate a design model and evaluate it The overall workflow involves the following steps: Generation of the TS ensemble Analysis of metal site in the PDB file(s) and classification Alignment of TS ensemble to the curated active site set Minimization of TS ensemble in a polyAla pocket (optional) Computational Redesign of Metalloenzymes 267 Fig (a) Scheme for the computational repurposing of active sites Different zinc coordination sites found in crystallographic structures in the Protein Databank are curated (e.g., tetrahedral and trigonal bipyramidal) and a model of the transition state of the reaction under consideration is superimposed on the open coordination site(s) of the metal ion in each PDB file LG is leaving group, and L1, L2 etc represent zinc ligands RosettaMatch and RosettaDesign are used to design additional TS stabilizing interactions (b) Using this approach, organophosphate hydrolysis activity (top) was designed into an adenosine deaminase (bottom) 268 Per Jr Greisen and Sagar D Khare Fig Example of computational enzyme repurposing (a) The original adenosine deaminase crystal structure bound to an inhibitor (PDB code 1A4L) (b) Structure of the active site after the new organophosphate hydrolase TS was superimposed (c) RosettaMatch was used to identify additional hydrogen bonds to the TS The residue Gln58 was placed to interact with the attacking nucleophile (d) RosettaDesign was used to identify additional TS stabilizing interactions The residue W65 was found to make pi–pi stacking interactions with the leaving group Introducing additional catalytic residues Sequence design to maximize TS affinity Reversion of destabilizing sequence changes to wild type identities Docking the TS models in the designed active site (validation) using RosettaDock and molecular dynamics simulations (Fig 3) Protein expression, purification, and experimental characterization (not discussed here) 2.1 Transition State Ensemble The TS ensemble is generated using a TS analog structure and/ or using quantum chemical simulations of the reaction under consideration For our purpose, we assume that a molecular model of the TS ensemble can be obtained For constructing the Rosetta model, typically we start from a molfile representation, and Computational Redesign of Metalloenzymes 269 Fig Design validation using docking RosettaDock was used to interrogate the energy landscape of the designed protein bound to the TS model A robust funnel indicated by low interface energies corresponding to the conformations similar to the designed position of the TS (low RMSD) suggests that alternative binding modes are disfavored convert it to Rosetta parameters using a script provided with the Rosetta software: /path/to/rosetta/rosetta_source/src/python/ apps/public/molfile_to_params.py 2.2 Analysis of Metal Site in the PDB File(s) and Classification by Coordination Geometry To select protein scaffolds suitable for design, all protein structures from the Protein Data Bank (PDB) are collected The search is limited to high-resolution structures (