Structure and dynamics studies of membrane and non-membrane proteins using NMR and MD simulations

The protein structural knowledge is essential in defining molecular recognition rules that power the understanding of basic biological phenomenon. The structures of most proteins are determinable due to advancement in technology and method development. Nuclear Magnetic Resonance (NMR) is one of the most versatile tools designed for this purpose. Proteins are flexible entities and dynamics play key role in their functionality therefore structures alone may provide only partial view on their functions. The experimental techniques have been used to study protein thermodynamics, but computer simulations have evolved to become the most convenient way to obtain the complete picture of protein dynamics. The central aim of this research is to study the structure and DNA binding dynamics of homologous pairing protein 2 (HOP2). In the first phase, the structure of N-terminal domain of HOP2 was investigated using NMR. It was identified with winged-helix DNA-recognition structural motifs. Furthermore, the DNA binding properties of this protein was investigated by NMR chemical shift perturbation method. It was found to bind to double-stranded DNA with considerable affinity, where structural motifs helix 3 (H3) and wing 1 (W1) were responsible for DNA recognition. Additionally, the site directed mutagenesis studies suggested H3 as the major contributor in DNA recognition. In the second phase, the DNA binding dynamics of HOP2 was investigated using classical MD simulations. Complexes of protein HOP2 and its mutants with DNA were constructed and then simulated using software GROMACS. Simulation results revealed the atomic level interactions between HOP2 and DNA, where H3 and W1 motifs engaged with DNA at major and minor grooves respectively. The effects on DNA binding due to point mutations in W1 and H3 were also observed. These effects were accessed in terms of changes in complex stability, binding free energy, and total number of interactions. The simulation results we obtained suggested that the motif W1 is also important as H3 in DNA binding. The NMR experimental and simulation protocol designed in this work will be useful in studying structure and dynamics of protein-protein or protein-ligand systems

Wing 1 of protein HOP2 is as important as helix 3 in DNA binding by MD simulation

<p>The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer’s foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.</p