The Structures And Dynamics Of The PDZ Domain From Protein LARG In Apo And Complex States

Rho GTPases play important roles in the cytoskeleton-involved cell processes such as cell migration,axon guidance,cell cycle events and membrane transport. Leukemia-associated Rho guanine nucleotide exchange factor(LARG)is a RhoA specific guanine nucleotide exchange factor(GEF)that can activate RhoA and may contribute to leukemogenesis.It consists of four domains:PDZ,RGS,DH and PH domains.The DH and PH domains directly contribute to the substrate catalysis and RhoA activation.The PDZ domain of LARG can interact with several membrane proteins,including plexin-B1,IGF-1 receptor and CD44.Through these interactions, extracellular signals can stimulate the activation of RhoA and be relayed to intracellular signals.The interaction of LARG PDZ with the carboxyl-terminal sequence of plexin-B1 promotes RhoA activation and thereby regulates cytoskeleton rearrangements during axonal and dendritic guidance,resulting in axonal growth cone collapse and cell migration.Several reports suggested that the interactions of the PDZ domain in RhoGEFs with diverse target receptors initiate conformational changes leading to the activation of RhoA GTPase.Until now there is no structural and dynamic information about these interactions.In this dissertation,we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide.Unobservable resonances of the residues inβB/βC andβE/αB loops in apo state were observed in the complex state.A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs.Major conformational changes in the LARG PDZ take place at the base of the binding groove,rather than the top portion as in case of other PDZs.At the same time,we detect the binding characteristics of the LARG PDZ domain with other ligands using the chemical shift perturbation experiments.Because of the unobservable resonance signals for the backbone amide groups in theβB/βC andβE/αB loops,we are interested in the dynamic properties of the LARG PDZ.Analysis of the ¹⁵N relaxation data using reduced spectral density mapping show that the apo LARG PDZ(especially its ligand-binding groove)is flexible and exhibit internal motions on both picosecond to nanosecond and microsecond to millisecond timescales.The CPMG relaxation dispersion experiment was used to quantify the chemical exchange on micro-to millisecond timescale and performed on LARG PDZ domain at different levels of saturation by the peptide VENKVTDL at two fields respectively.At the apo state,mapping of the residues with slow internal motions onto the solution structure revealed that these residues are clustered in the ligand binding side.In the low saturation state(the concentration ratio of the protein to peptide is 10:1),the conformational exchange contribution(R_ex)to the transverse relaxation rate is more obvious and larger.The conformational exchange rates are reduced.In the complex form,the steric interactions,hydrogen bonds and electrostatic interactions dramatically reduced the internal motions upon binding of the plexin B1 peptide.Considering the structures and the dynamic properties of the LARG PDZ,we are curious about the correlation between the structures and dynamics.Mutagenesis and thermodynamic studies indicate that the conformation of theβB/βC andβE/αB loops affects the PDZ-peptide interaction.It is suggested that the conformation of theβB/βC loop andβE/αB loop affects the binding characteristics of the LARG PDZ. These two loops with fast and slow internal motions are apt to work in a cooperative way to drive the reorientation ofβB andαB to widen the binding groove and accommodate the ligands.In the LARG PDZ domain,the loop mobility may contribute to the conformational change of the binding groove.The conformational flexibility could facilitate the changes in structures upon the binding of different ligands.