Molecular Simulation Approaches to Proteins Structure and Dynamics and to Ligand Design

Molecular simulations approaches are powerful tools for structural biology and drug discovery. They provide additional and complementary information on structure, dynamics and energetics of biomolecules whose structures have been determined experimentally [1,2,3,4,5,6,7]. In particular, Molecular Dynamics (MD) simulations [8], along with elastic network analysis [9], offer insights into molecular fluctuations, conformational changes and allosteric mechanisms. In addition, molecular simulation can be used to design novel and potent ligands to a specific target (either a protein or DNA) as well as to estimate ligands potency [10,11]. Attempts at predicting protein structures using bioinformatics and MD are also increasingly successful [12,13,14,15], as well as approaches that use solely simulation tools [16]. The development of new algorithms and the continuously growing computer power currently allow for the simulation of more and more complex biological systems, such as protein aggregates [7,17,18,19,20] and protein/DNA complexes [21]. In this context, a number of theoretical techniques (namely molecular dynamics simulations, elastic network analysis, electrostatic modeling and binding energy predictions) have here been applied to the study of specific proteins. On the basis of X-ray protein structures, molecular simulations have provided a detailed description of internal motions and interactions, which are not evident from the experimental data and have functional implications. First, we have used MD to investigate structural features, focusing on the differences between the solid state and the aqueous solution structures. Over 80% of data in the PDB [22] are X-ray structures, making protein crystallography the major resource in structural biology. Nevertheless, in a few cases, the structural details might be affected by environmental features, such as the presence of small compounds in the buffering solution and/or crystal packing contacts due to the periodic lattice. Here, a comparative MD study has been performed on the Catabolite Activator Protein (CAP), in both the crystal phase and in the aqueous solution. CAP is a bacterial DNA-binding transcription regulator whose activity is controlled by the binding of the intracellular mediator cyclic Adenosine MonoPhosphate (cAMP). CAP is a homodimeric protein and each subunit is formed by a cyclic nucleotide- and a DNA-binding domain. Inspection of the available CAP X-ray structure within the crystal environment [23] suggests that packing contacts do affect the native conformation of the ligand activated protein. Anticipating our results, we have found that indeed the conformation of the protein in solution is different, and that these differences may play a role for CAP biological function. Next, we have used molecular simulations to target structural flexibility. Conformational fluctuations often play a key role for the protein function and MD simulations can provide information on large-scale concerted motions of proteins [24,25,26]. We have addressed this point in the context of the Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) cation channel. The tetrameric HCN channels are opened by membrane hyperpolarization, while their activation is allosterically modulated by the binding of cAMP in the cytoplasm. The cytoplasmic part of the HCN2 channel, which is responsible for the channel modulation, has been here investigated by MD simulations and elastic network analysis, on the basis of the available X-ray structure [27], to earn new insights into the molecular mechanism triggered by cAMP. We have found that, in the presence of cAMP, the protein undergoes a quaternary structure oscillation, in which each subunit moves as a rigid body. This fluctuation, which is not observed in the absence of cAMP, could facilitate the channel opening transition. Finally, we have moved our attention to an issue relevant for structure-based drug design. Within a long-standing collaboration with Prof. Cattaneo’s lab (SISSA and Motivations and Summary 7 Layline Genomics), our group has been interested in the design of mimics of proteins involved in the biochemical pathways that lead to the Alzheimer’s disease. Here, on the basis of structural information [28], we have designed a peptide that could specifically target trkA, the high affinity receptor of the Nerve Growth Factor (NGF), which is a protein that plays a critical role for the development, survival and maintenance of neurons in the vertebrate nervous system and activates signaling pathways related to neuroprotection. The results of this research will be tested at the Prof. Cattaneo’s Lab in order to validate the theoretical findings and assess the potency and the effects of such a ligand.