Using Molecular Dynamics Simulations To Provide New Insights into Protein Structure on the Nanosecond Timescale:  Comparison with Experimental Data and Biological Inferences for the Hyaluronan-Binding Link Module of TSG-6

Abstract

Link module domains play an essential role in extracellular matrix assembly and remodeling by binding to the flexible glycosaminoglycan hyaluronan. A high-resolution NMR-structure of the Link module from the protein product of tumor necrosis factor-stimulated gene-6 (Link_TSG6) has been determined, but a fuller appreciation of protein dynamics may be necessary to understand its hyaluronan-binding. Therefore, we have performed a 0.25 μs MD simulation, starting from the lowest-energy NMR-derived solution structure of Link_TSG6, with explicit water and ions, using the CHARMM22 protein force field. The simulation was as good a fit to the NMR data as the ensemble from simulated annealing, except in the β5-β6 loop. Furthermore, analysis revealed that secondary structure elements extended further than previously reported and underwent fast picosecond time scale dynamics, whereas nanosecond dynamics was found in certain loops. In particular, surface side chains proposed to interact with glycosaminoglycans were predicted to be highly mobile and be directed away from the protein surface. Furthermore, the hyaluronan-binding β4-β5 loop remained in a closed conformation, favoring an allosteric interaction mechanism. This enhanced view of the Link module provides general insight into protein dynamics and may be helpful for understanding the dynamic molecular basis of tissue assembly, remodeling, and disease processes.

Structure and dynamics of the kinase IKK-β – A key regulator of the NF-kappa B transcription factor

“…In order to identify small-scale conformational changes and novel allosteric binding sites the model was subjected to constant temperature MD simulations over 80 ns. While simulations of this time scale are too short to reveal large conformational changes or even protein folding events, it was shown that this time scale is sufficient to explore the conformation of loops and side-chains in agreement with experimental data (Almond et al, 2007;Nederveen and Bonvin, 2005). Our results reveal the molecular details of ATP and peptide substrate ligand binding, the role of the activation loop and conformational changes.…”

“…The apo-form showed significant structural changes throughout, which would be expected as the protein structure was modelled with the ATP cofactor bound. It should be noted that the simulation time of 80 ns limits the conformational changes to small scale motions in the vicinity of the starting structure, which nevertheless allow the exploration of side-chain movements and loop flexibility that is in agreement with NMR experiments (Almond et al, 2007) and may critically influence ligand binding.…”

The molecular basis of inter-α-inhibitor heavy chain transfer on to hyaluronan

“…Conversely, our studies showed that a change in the ionization state of the Link TSG6 residue His 45 is largely responsible for the gain in HA affinity between pH 3.5 and 6; His 45 (equivalent to His 80 in full-length TSG-6) lies adjacent to the HA-binding site and in close proximity to the β4-β5 loop that undergoes a conformational change on association with HA. Another recent development in our investigation of the Link TSG6/HA interaction has come from the use of Molecular Dynamics simulations, allowing insights into the structure of this protein on a nanosecond timescale [9]. The data obtained supported our previous conclusion of an induced-fit mechanism for HA binding TSG-6 (with Link and CUB modules indicated by L and C respectively) reacts with IαI to form covalent TSG-6·HC complexes.…”

Plasticity of the TSG-6 HA-binding Loop and Mobility in the TSG-6-HA Complex Revealed by NMR and X-ray Crystallography

“…48 Correlation times, τ c , were extracted following the procedure described by Farrow et al 49 Comparisons of the T 1 , T 2 and heteronuclear NOE data for Link_TSG6 with molecular dynamics simulations of the free protein have been documented previously. 50 Crystallography Crystals were grown using the sitting-drop method. A 2.5 μl drop formed from equal amounts of protein solution (7 mg/ml solution in approximately 25 mM Tris-HCl, 75 mM NaCl) and well solution was equilibrated against 1.0 ml well solution (2 M ammonium sulphate, 2% (v/v) PEG 400, 0.1 M sodium Hepes (pH 7.5)).…”