Study On The Structure-Function Relationship Of Proteins Of The Periplasmic Transport System By Molecular Modeling Methods

In this thesis,the molecular modeling methods have been applied to the glutamine binding protein (GlnBP), which has the typical structure of periplasmic binding protein (PBP), with the aim to understand the functional motion, substrate-capture/-release mechanisms of PBP. Since it is difficult to obtain the crystal structure of the membrane binding component (MBC) of the glutamine transport system, the molecular dynamics simulation (MD) has been applied to BtuCD, which is the MBC of the vitamine B12 transport system with its crystal structure available, with the aim to observe the intrinsic functional motions and understand the transmembrane substrate-translocation mechanism of MBC. The content of the thesis comprises the following major aspects:(1) Study on the structure-function relationship of GlnBP and its relationship with substrate by molecular modeling methodBasing on the crystal structures of GlnBP in substrate-free open form and in glutamine (Gln) -bound close form, MD simulation method and docking method have been applied to study the intrinsic functional motions of GlnBP, structure characteristics of GlnBP-Gln complex, the interaction between GlnBP and Gln and the binding mode between Gln and GlnBP in ligand-free open form.The results of MD simulations show that there exist significant inter-domain motions in GlnBP, i.e. an open-close motion and a twisting motion. A region consisting of residue Lys115-Gly147 which moves as a whole mainly contributes such an open-close motion and a large loop of residue Ala96-Lys110 that is adjacent this region confers the flexibility of such a motion. The hinge region between the two domains exhibits surprisingly rigidity. In Gln-bound close form, the inter-domain motions of GlnBP are restricted and the structure of the GlnBP-Gln complex is rather stable. The close conformation of GlnBP is maintained mainly by the network of hydrogen bond involving Gln.The binding modes between GlnBP in open form and Gln as demonstrated by the docking method show that Gln may bind to GlnBP at a binding site in the active pocket near the small domain at first, and then begin the conformation transition to close form. The intrinsic inter-domain motions and the forming of the hydrogen-bond pairs may drive the process of substrate capture. These results may help to understand the substrate-capture mechanism of PBP in periplasmic transport system.(2) Collective motion directed molecular dynamics simulationIn order to further understand the substrate-capture mechanism of GlnBP, it is necessary to enhance the inter-domain motions of GlnBP during MD simulation. Here the anisotropy network model (ANM) analysis method was incorporated into the classical MD simulation by using ANM to indicate the directions of the collection modes and apply external forces in these directions during MD simulation, with the aim to enhance the functional motions of a protein.Such a modified MD simulation method was applied to a pentapeptide and GlnBP in open form. The primary results show that the conformation transition and functional motions of the proteins are enhanced. Further improvement should be made on this method and then applied it on the GlnBP to study the substrate-capture mechanism.(3) Ligand release pathway of GlnBP as revealed by using steered molecular dynamics simulation methodSteered molecular dynamics (SMD) simulation method has been employed to lead the substrate Gln out of the binding pocket of GlnBP to reveal the substrate release pathway and the interaction sites of GlnBP with its corresponding MBC.The results of the SMD simulations show that a novel back-door pathway other than a front-door pathway is more preferable for the substrate release. The potential of mean forces (PMFs) reconstructed from multiple SMD trajectories indicate that the potential barrier for release along the back-door pathway is lower than along the front-door pathway. Additionally, the structural characteristics and the electrostatic characteristics of the surface of GlnBP support this result. There are four negative-charged residues locating around the exit of the back-door pathway, which may involve in the"docking"of the ligand release channel of GlnBP with the transmembrane channel in its corresponding positive-charge–rich MBC. Normal mode analysis (NMA) suggested that the intrinsic functional motions of GlnBP-Gln complex can enlarge the back-door pathway and make substrate release much easier.This work may help to understand the substrate-release mechanism of the periplasmic transfer system and additionally give some clues to study how the substrate-bound complex binds to its corresponding MBC and completes the subsequent translocation of the substrate.(4) Molecular dynamics simulation on BtuC embedding in the POPC lipid bilayerBtuC was inserted into a palmitoyl oleoyl phosphatidylcholine (POPC) lipid bilayer, and MD simulation method was applied on the BtuC-POPC system to study the functional motions of BtuC.A stable BtuC-POPC system was obtained by a more-than-57-nanosecond MD simulation. It was found that the POPC lipid bilayer had the ability to adjust its thickness to match with the BtuC embedding in it. The area per lipid and the bilayer thickness, which are the two parameters characterizing a lipid bilayer, are in agree well with the experiment results of POPC lipid bilayer. The main functional motions of BtuC were demonstrated on the atomic level by applying the principle component analysis (PCA) on the trajectories of the MD simulation. The results show that all the main functional motions are different, but the effect of them is to change the dimension of the transmembrane channel and control the open and close of the gate of the channel. These motions of BtuC are compatible with the functional motions of the two components which couple with BtuC, i.e. BtuF and BtuD. The motions of BtuC are mainly located at the regions in the periplasmic space, and control the dimension of the channel at this side. At the cytoplasmic side, the dimension of the channel did not exhibit significant change. Surprisingly, the motions of the two domains of BtuC are different though they share the same sequence and similar advanced structure. The study on the functional motions of BtuC can help to understand the transmembrane substrate-translocation mechanism of periplasmic transport system.