Functional Domain Motions and Processivity in Bacterial Hyaluronate Lyase: A Molecular Dynamics study Dissertation Zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakult¨aten der Georg-August-Universit¨at zu G¨ottingen Vorgelegt von Harshad Joshi aus Pune, Indien G¨ottingen, 2007 D7 Referent: Prof. Dr. Tim Salditt Korreferent: PD Dr. Helmut Grubmuller¨ Tag der mundlichen¨ Prufung:¨ To my parents and teachers Great things can be achieved by putting small things together. Just like a rope made of grass can be used to tie down a massive mad elephant. (In the hope that this thesis contributes in better understanding of the mother nature.) Contents 0 Prologue xi 0.1 Scope of the thesis . xiii 0.2 Outlineofthethesis .............................. xvi 1 Theory and Methods 1 1.1 Molecular Dynamics simulations - Principle . 3 1.1.1 From Schr¨odinger equation to Molecular Dynamics . 4 1.2 MD simulations in practice . 9 1.2.1 Integration method and time step . 9 1.2.2 Solvent environment . 10 1.2.3 System boundaries . 11 1.2.4 Temperature and pressure coupling . 12 1.2.5 Improving efficiency . 14 1.2.6 Minimisation and equilibration . 16 1.3 Analysis methods . 17 1.3.1 Principal Component Analysis . 17 2 Processivity and Hyaluronidases 21 2.1 Processive Enzymes . 23 2.2 Hyaluronidases . 25 2.2.1 History and physiological importance . 26 2.2.2 Classification of Hyaluronidases and Spn.Hyaluronate Lyase . 27 2.3 Streptococcus Pneumoniae Hyaluronidase (Spn.Hyal) . 28 2.3.1 Choice of Spn.Hyal for processivity study . 29 2.3.2 Structure of Spn.Hyal and its catalytic mechanism . 29 2.3.3 Previous studies on the processive mechanism of Spn.Hyals . 31 i 3 Hyaluronan: Structure and dynamics 35 3.1 Hyaluronan (HA): Introduction . 37 3.1.1 Chemical structure and polymer properties . 37 3.1.2 Solution structure and aggregation properties . 38 3.1.3 Occurance and physiological importance . 39 3.1.4 Medical applications . 41 3.2 Force field choice . 41 3.2.1 Simulation setup . 42 3.2.2 Validation and choice of OPLS-SEI* force field . 43 3.2.3 Flexibility of HA in the presence of the Spn.Hyal . 45 3.3 Conclusions . 46 4 Flexibility of bacterial Hyals: Part I 49 4.1 Conformational motions of proteins . 51 4.2 Crystallisation of new Spn.Hyal structures . 52 4.2.1 Crystallisation and diffraction data collection . 52 4.2.2 Structure solution and refinement . 52 4.3 Simulation details . 53 4.4 Results . 55 4.4.1 Description of the new pneumococcal Hyal structures . 55 4.4.2 Bacterial Hyals are flexible . 55 4.5 Discussion . 59 4.6 Conclusions . 61 5 Flexibility of bacterial Hyals: Part II 63 5.1 Flexibility of Spn.Hyaluronate Lyases . 65 5.2 Simulation details . 66 5.3 Results and Discussions . 67 5.3.1 Equilibration and the flexibility of the Spn.Hyals . 68 5.3.2 PCA and the domain motions in Spn.Hyals . 69 5.3.3 Flexibility of the HA inside the cleft . 75 5.3.4 Processive motion of the sugar . 77 5.3.5 Role of protein dynamics in substrate binding . 79 5.3.6 Interaction of the HA with the positive and aromatic patch of the cleft ................................... 81 5.3.7 Characterisation of important known residues . 84 5.3.8 Other Interactions . 87 5.3.9 Complementary forces and origin of an energy barrier . 87 5.4 Conclusions . 89 6 Force-induced MD simulations of Spn.Hyal-HA system 91 6.1 Kinetic traps and force-induced simulations . 93 6.2 Elevated temperature simulations . 94 6.2.1 Principle ................................ 94 6.2.2 Simulation setup . 95 6.2.3 Results and Discussions . 95 6.2.4 Conclusions . 98 6.3 Force Probe Molecular Dynamics (FPMD) simulations: Principle . 99 6.4 Essential Dynamics (ED) Sampling: Principle . 101 6.5 FPMD simulations: Results and discussions . 102 6.5.1 Rupture force, pulling speed and force constant . 102 6.5.2 Protein flexibility in FPMD simulations . 104 6.6 ED simulations: Results and discussions . 109 6.7 Free energies . 112 6.7.1 Free energies from equilibrium trajectories . 112 6.7.2 Free energy profile for HA translocation . 113 6.7.3 Free energies from non-equilibrium trajectories . 116 6.8 Conclusions . 116 7 Epilogue 119 7.1 what remains to be done... 123 8 Musings 125 A Abbreviations used 127 B HA force field parameters 129 C Principle of AFM experiments 133 List of Figures 1 Schematic representation of simulation method dependence on the size of the system and the timespan of the process of interest. xiii 2 A typical Spn.Hyal-HA simulations setup. xv 1.1 Illustration of the force field terems. 7 1.2 Typical molecular dynamics simulation system with explicit solvent box. 13 1.3 A schematic representation of Principal Component Analysis (PCA) for the MD-trajectory. 18 2.1 Representative structures for each class of processive enzymes. 25 2.2 Schematic diagram of the virulence factors of Strep. Pneumoniae...... 26 2.3 Structural details of Spn.Hyal. with the electrostatic potential distribu- tion in the catalytic cleft. 30 2.4 Details of the catalytic mechanism in Spn.Hyal-HA system. 32 2.5 The three largest domain motions of Spn.Hyal as obtained by CONCOORD. 33 3.1 Chemical structure of Hyaluronic acid. 37 3.2 Solution structure of Hyaluronic acid I — Hydrogen bonding. 39 3.3 Solution structure of Hyaluronic acid II — Aggregation properties. 40 3.4 2D projections of simulations with different force fields on the PCA eigenvector- set of experimental structures. 44 3.5 Dihedral distribution along the glycosidic linkages of HA6 polymer for the simulations with different force fields. 45 3.6 Difference in dihedral distribution along the glycosidic linkages of HA6 polymer in the presence or absence of the Spn.Hyal. 46 4.1 A schematic representation of the relationship of the flexibility of the protein and the energy landscape explored by it. 51 v 4.2 Difference in the extent of the opening/closing of the catalytic cleft for different crystal structures discussed in the chapter. 56 4.3 MD simulation trajectories show that the flexibility of the Spn.Hyal as affected by the presence of the HA hexasaccharide inside the cleft. Simula- tion with holo structure remains ‘locked’ while the cleft opens significantly in the apo-simulation. 57 5.1 Root mean square fluctuations (RMSF) per residue for the simulations show the three loop regions around the cleft of the Spn.Hyal are highly flexible. ..................................... 68 5.2 2-D Projections of the simulation trajectories together with the x-ray structures projected on the essential subspace obtained from the PCA. These projections show the flexibility of the protein along the three do- main motions observed in Chapter 4 . 70 5.3 Time evolution of the simulation trajectories together with the x-ray structures projected on the essential subspace obtained from the PCA. 71 5.4 Motion of the sugar inside the cleft measured as the distance from the catalytic cleft. 76 5.5 Flexibility of the sugar inside the cleft measured as the root mean square deviation (RMSD) of the first four rings of HA with respect to those in the crystal structure 1LOH.pdb . 77 5.6 Motion of the sugar in the processive direction inside the cleft as depicted by the PCA performed on the sugar. 78 5.7 Short range interactions of the sugar with the positive and the aromatic region in the cleft. 82 5.8 Spn.Hyal-HA interface region showing important residues that are in con- tact with the HA in the crystal structure 1LOH.pdb . 84 5.9 Short range interactions of the individual sugar rings with the specific residues that are known from the mutation experiments or are thought to play a key role in the processivity mechanism. 86 5.10 General regions around the catalytic cleft. 88 5.11 Short range interactions of the last disaccharide units (at non-reducing end) of the HA with the entry region of the cleft. 89 6.1 Schematic representation of the energy landscapes explored by the system at different temperatures. 94 6.2 2-D & 1-D Projections of the simulation trajectories together with the x-ray structures projected on the essential subspace obtained from the PCA. The increased temperature of the sugar affected the flexibility of the protein indicating strong coupling of the protein dynamics to the sugar mobility. 96 6.3 Flexibility of the sugar inside the cleft for the high-temperature simula- tions as measured from the RMSD and the PCA of the first four rings of thesugar. .................................... 98 6.4 A typical Force Probe Molecular Dynamics (FPMD) simulation for Spn.Hyal- HA system. 100 6.5 Schematic representation of Essential Dynamics technique. 101 6.6 FPMD simulation set up for the Spn.Hyal-HA system. 102 6.7 Effect of different force constants and pulling velocities on the rupture forces. 105 6.8 Scheme of the FPMD simulations setup to investigate the protein flexi- bility during the sugar translocation. 106 6.9 Relationship between the protein flexibility and the sugar translocation through the cleft. 107 6.10 Rupture forces for different FPMD simulations and a probable explaina- tion for the easier pulling in the opposite direction. 108 6.11 Short range interactions of the sugar with the cleft of the Spn.Hyal in different EDSAM simulations. 111 6.12 Free energy estimates for the sugar translocation along the putative re- action pathway from different enforced simulations. 114 B.1 Schematic representation of HA disaccharide unit with the atom numbers labeled for the topology.