Periodic Reporting for period 1 - DynaPLIX (Dynamics of Protein–Ligand Interactions)
Reporting period: 2023-05-01 to 2024-10-31
Proteins are vital to all processes of life. Understanding the functions of proteins has great scientific and commercial value: proteins are used as industrial enzymes, as pharmaceutical treatments, and many proteins are the targets of drugs. Current knowledge of protein function is primarily based on static structuresstructures, which have provided great insights into structure-function relationships that form the basis for protein science and protein engineering. However, proteins are not static molecules, but undergo spontaneous transitions between alternative structural states, some of which are transient conformations essentially invisible to traditional methods. These dynamical properties are known to be critically important for protein function, but high-resolution studies of dynamics have so far been conducted merely as an “add-on” following structural studies.
To change the situation, we aim to establish “integrative biomolecular dynamics” by developing methods that integrate time-resolved X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular simulations to study the motions of proteins while they carry out their function. We focus on the challenging problem of molecular recognition because it represents a poorly understood frontier in molecular science where advances are expected to have great impact. Specifically, we will address the question of how proteins bind ligands by describing with atomic resolution the entire dynamic process to reach a consistent kinetic, thermodynamic, and structural view.
We are at a point where it will be possible to develop the individual techniques required for our integrative biomolecular dynamics approach. As a team we can leverage ongoing developments in hardware and methods, while ensuring the tight integration between methods that is needed to study complex dynamical systems. We thus aim to move structural biology into a new era of protein dynamics.
To change the situation, we aim to establish “integrative biomolecular dynamics” by developing methods that integrate time-resolved X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular simulations to study the motions of proteins while they carry out their function. We focus on the challenging problem of molecular recognition because it represents a poorly understood frontier in molecular science where advances are expected to have great impact. Specifically, we will address the question of how proteins bind ligands by describing with atomic resolution the entire dynamic process to reach a consistent kinetic, thermodynamic, and structural view.
We are at a point where it will be possible to develop the individual techniques required for our integrative biomolecular dynamics approach. As a team we can leverage ongoing developments in hardware and methods, while ensuring the tight integration between methods that is needed to study complex dynamical systems. We thus aim to move structural biology into a new era of protein dynamics.
WP1, Protein expression and purification
Established recombinant over-expression of soluble protein for 4 model systems: Klebsiella pneumoniae CTX-M-14, bacteriophage T4 lysozyme (T4L), HIV protease, and Helicobacter pylori MurI. Successfully purified T4L wildtype, T4L L99A mutant, as well as CTX-M-14 and its activity-impaired mutants S70A, S70G, and E166A. T4L L99A and CTX-M-14 wildtype have also been produced in isotopically (15N, 13C, 2H) labeled forms for NMR experiments. Purification of the other model systems is currently in progress.
WP2, Protein crystallization
Protein crystallization achieved for CTX-M-14, T4L, as well as Human Insulin (HI), which serves as an additional, commercially available model system. For all systems, crystallization protocols have been optimized to either yield well-diffracting macro-crystals or highly homogenous micro-crystals of a specific size to streamline time-resolved experiments.
WP3, Static structure determination
Crystal structures determined for CTX-M-14 (up to 0.77 Å), for T4L (up to 0.85 Å), and HI (up to 1.3 Å) in ligand-free and ligand-bound forms, both at cryogenic and ambient temperatures. These structures serve as important reference endpoints of the ligand binding pathways for comparison with TR-SSX data.
WP4, TR-SSX
Cryo-trapping as well as TR-SSX approaches employed for CTX-M-14, T4L, as well as HI. The binding of several ligands to T4L L99A was systematically tested (indole, 5-bromoindole, 7-bromoindole, 5,7-dibromoindole). The endpoint of indole binding to T4L was obtained. To normalize ligand occupancy as a function of time, two different T4L L99A crystal sizes (30 µm vs. 15 µm) were probed. A stable endpoint of ligand binding was obtained for HI via cryo-trapping crystallography, demonstrating sulfate binding within 250 ms.
WP5, Isothermal titration calorimetry
Annual service of the instrument, followed by initial test experiments to validate its operational capacity.
WP6, NMR relaxation experiments
Validated chemical shift assignments of T4L L99A in its apo state as well as CTX-M-14 in both its apo and tazobactam-bound states. Acquired NMR relaxation data on these states at two static magnetic field strengths (14.1 T and 18.8 T) and at three temperatures (10, 20, and 30 °C for T4L L99A; 20, 30, and 37°C for CTX-M-14).
WP7, NMR relaxation methods development
Initiated the design and implementation of CEST experiments for aromatic 13C nuclei, in order to study exchange of aromatic ligands between the free and bound states, as well as conformational dynamics of aromatic side chains in proteins.
WP8, Methods for analyzing time-dependent NMR data
Initiated and made substantial progress in developing the algorithms and software needed to integrate time-dependent NMR experiments, notably NMR relaxation dispersion measurements.
WP9, Methods for analyzing time-resolved data
Developed an approach for modeling non-equilibrium dynamics on free energy surfaces constructed by molecular dynamics simulations, and calculated long-timescale relaxation dynamics on these. This work differs from that in WP8 in that this is aimed to model non-equilibrium processes such as those probed by TR-SSX in WP4.
WP10, Simulation studies of Mpro; WP11, Simulation studies of MurI and HIV PR
Performed extensive simulations of CTX-M-14 and T4L L99A. Modeling the temperature-dependent dynamics of CTX-M-14. Performed extensive simulations of ligand (benzene) binding to T4L, both to test models for sampling ligand binding processes and for interpreting them (WP8 & WP9).
Established recombinant over-expression of soluble protein for 4 model systems: Klebsiella pneumoniae CTX-M-14, bacteriophage T4 lysozyme (T4L), HIV protease, and Helicobacter pylori MurI. Successfully purified T4L wildtype, T4L L99A mutant, as well as CTX-M-14 and its activity-impaired mutants S70A, S70G, and E166A. T4L L99A and CTX-M-14 wildtype have also been produced in isotopically (15N, 13C, 2H) labeled forms for NMR experiments. Purification of the other model systems is currently in progress.
WP2, Protein crystallization
Protein crystallization achieved for CTX-M-14, T4L, as well as Human Insulin (HI), which serves as an additional, commercially available model system. For all systems, crystallization protocols have been optimized to either yield well-diffracting macro-crystals or highly homogenous micro-crystals of a specific size to streamline time-resolved experiments.
WP3, Static structure determination
Crystal structures determined for CTX-M-14 (up to 0.77 Å), for T4L (up to 0.85 Å), and HI (up to 1.3 Å) in ligand-free and ligand-bound forms, both at cryogenic and ambient temperatures. These structures serve as important reference endpoints of the ligand binding pathways for comparison with TR-SSX data.
WP4, TR-SSX
Cryo-trapping as well as TR-SSX approaches employed for CTX-M-14, T4L, as well as HI. The binding of several ligands to T4L L99A was systematically tested (indole, 5-bromoindole, 7-bromoindole, 5,7-dibromoindole). The endpoint of indole binding to T4L was obtained. To normalize ligand occupancy as a function of time, two different T4L L99A crystal sizes (30 µm vs. 15 µm) were probed. A stable endpoint of ligand binding was obtained for HI via cryo-trapping crystallography, demonstrating sulfate binding within 250 ms.
WP5, Isothermal titration calorimetry
Annual service of the instrument, followed by initial test experiments to validate its operational capacity.
WP6, NMR relaxation experiments
Validated chemical shift assignments of T4L L99A in its apo state as well as CTX-M-14 in both its apo and tazobactam-bound states. Acquired NMR relaxation data on these states at two static magnetic field strengths (14.1 T and 18.8 T) and at three temperatures (10, 20, and 30 °C for T4L L99A; 20, 30, and 37°C for CTX-M-14).
WP7, NMR relaxation methods development
Initiated the design and implementation of CEST experiments for aromatic 13C nuclei, in order to study exchange of aromatic ligands between the free and bound states, as well as conformational dynamics of aromatic side chains in proteins.
WP8, Methods for analyzing time-dependent NMR data
Initiated and made substantial progress in developing the algorithms and software needed to integrate time-dependent NMR experiments, notably NMR relaxation dispersion measurements.
WP9, Methods for analyzing time-resolved data
Developed an approach for modeling non-equilibrium dynamics on free energy surfaces constructed by molecular dynamics simulations, and calculated long-timescale relaxation dynamics on these. This work differs from that in WP8 in that this is aimed to model non-equilibrium processes such as those probed by TR-SSX in WP4.
WP10, Simulation studies of Mpro; WP11, Simulation studies of MurI and HIV PR
Performed extensive simulations of CTX-M-14 and T4L L99A. Modeling the temperature-dependent dynamics of CTX-M-14. Performed extensive simulations of ligand (benzene) binding to T4L, both to test models for sampling ligand binding processes and for interpreting them (WP8 & WP9).
Resolving ligand-binding processes based on NMR relaxation data.
We demonstrated that NMR relaxation dispersion data, acquired as a function of ligand concentration, can be analyzed to resolve induced-fit (IF) and conformational-selection (CS) pathways of protein–ligand complex formation. We obtained an essentially complete description of all rate constants characterizing the 4-step binding equilibria involving the IF and CS pathways. Notably, the flux of ligand binding is totally dominated by the IF pathway despite the binding affinity of the initial step of the CS pathway being more than 200-fold higher than that of the IF pathway. This is explained by the fact that the ligand achieves transition-state stabilization of the conformational change of the protein. Next we aim to perform experiments addressing the nature of encounter complexes.
Modeling NMR relaxation experiments
We developed approaches to perform simulations on free energy surfaces constructed by molecular dynamics simulations and to calculate long-timescale dynamics and NMR properties from these. Specifically, we implemented an approach in which we (i) perform enhanced sampling simulations, (ii) construct free energy surfaces from these, and (iii) perform additional dynamics as diffusive (Langevin) dynamics on these free energy surfaces. Using these dynamics, we then construct models of long-timescale dynamics and NMR chemical shift modulations from which we can calculate NMR relaxation-dispersion data. This is an ambitious, high-risk approach that we believe will transform our ability to understand protein dynamics. Much work remains to be done, but initial work looks promising.
We demonstrated that NMR relaxation dispersion data, acquired as a function of ligand concentration, can be analyzed to resolve induced-fit (IF) and conformational-selection (CS) pathways of protein–ligand complex formation. We obtained an essentially complete description of all rate constants characterizing the 4-step binding equilibria involving the IF and CS pathways. Notably, the flux of ligand binding is totally dominated by the IF pathway despite the binding affinity of the initial step of the CS pathway being more than 200-fold higher than that of the IF pathway. This is explained by the fact that the ligand achieves transition-state stabilization of the conformational change of the protein. Next we aim to perform experiments addressing the nature of encounter complexes.
Modeling NMR relaxation experiments
We developed approaches to perform simulations on free energy surfaces constructed by molecular dynamics simulations and to calculate long-timescale dynamics and NMR properties from these. Specifically, we implemented an approach in which we (i) perform enhanced sampling simulations, (ii) construct free energy surfaces from these, and (iii) perform additional dynamics as diffusive (Langevin) dynamics on these free energy surfaces. Using these dynamics, we then construct models of long-timescale dynamics and NMR chemical shift modulations from which we can calculate NMR relaxation-dispersion data. This is an ambitious, high-risk approach that we believe will transform our ability to understand protein dynamics. Much work remains to be done, but initial work looks promising.