Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites

Binding Site Identification and Flexible Docking of Single Stranded RNA to Proteins Using a Fragment-Based Approach

<div><p>Protein-RNA docking is hampered by the high flexibility of RNA, and particularly single-stranded RNA (ssRNA). Yet, ssRNA regions typically carry the specificity of protein recognition. The lack of methodology for modeling such regions limits the accuracy of current protein-RNA docking methods. We developed a fragment-based approach to model protein-bound ssRNA, based on the structure of the protein and the sequence of the RNA, without any prior knowledge of the RNA binding site or the RNA structure. The conformational diversity of each fragment is sampled by an exhaustive RNA fragment library that was created from all the existing experimental structures of protein-ssRNA complexes. A systematic and detailed analysis of fragment-based ssRNA docking was performed which constitutes a proof-of-principle for the fragment-based approach. The method was tested on two 8-homo-nucleotide ssRNA-protein complexes and was able to identify the binding site on the protein within 10 Å. Moreover, a structure of each bound ssRNA could be generated in close agreement with the crystal structure with a mean deviation of ~1.5 Å except for a terminal nucleotide. This is the first time a bound ssRNA could be modeled from sequence with high precision.</p></div

Comparison to the bound form of the poses obtained by (I) bound docking, (II) position-specific and (III) non-position specific filtering of chain-forming poses.

<p>Comparison to the bound form of the poses obtained by (I) bound docking, (II) position-specific and (III) non-position specific filtering of chain-forming poses.</p

Best approximation of the RNA bound form obtained by biased fragment docking and chain-propensity filtering.

<p>The bound form of the RNA in 1B7F (A) or 1CVJ (B) is represented as white sticks, and the best approximation for each fragment as lines, colored from blue to orange by green for frag1 to frag6. In both cases, frag5 and frag6 are best approximated by the same docking pose.</p

Flowchart illustrating the strategy for protein-ssRNA docking with structural fragments.

<p>Flowchart illustrating the strategy for protein-ssRNA docking with structural fragments.</p

Comparison to the bound form of the poses obtained by (I) unbound docking, then (II) chain-propensity filtering, then (III) 3Å-clustering and chain-propensity filtering.

<p>Comparison to the bound form of the poses obtained by (I) unbound docking, then (II) chain-propensity filtering, then (III) 3Å-clustering and chain-propensity filtering.</p

Structures of ssRNA-protein test-cases complexes.

<p>(A) Crystallographic structure of the human sex-lethal protein bound to a 5'-U8 ssRNA, PDB ID 1B7F. (B): Crystallographic structure of the polyA-binding protein bound to a 5'-A8 ssRNA, PDB ID 1CVJ. The protein is represented in gray cartoon, the RNA in stick colored as rainbow from blue (5', frag1) to red (3', frag6). The nucleotides discarded for docking experiments are colored in white.</p