Balasubramanian Chandramouli

Understanding the role of dynamics in the iron sulfur cluster molecular machine

Background The bacterial proteins IscS, IscU and CyaY, the bacterial orthologue of frataxin, play an essential role in the biological machine that assembles the prosthetic Fe—S cluster groups on proteins. They form functionally binary and ternary complexes both in vivo and in vitro. Yet, the mechanism by which they work remains unclear. Methods We carried out extensive molecular dynamics simulations to understand the nature of their interactions and the role of dynamics starting from the crystal structure of a IscS-IscU complex and the experimentally-based model of a ternary IscS-IscU-CyaY complex and used nuclear magnetic resonance to experimentally test the interface. Results We show that, while being firmly anchored to IscS, IscU has a pivotal motion around the interface. Our results also describe how the catalytic loop of IscS can flip conformation to allow Fe—S cluster assembly. This motion is hampered in the ternary complex explaining its inhibitory properties in cluster formation. Conclusions We conclude that the observed ‘fluid’ IscS-IscU interface provides the binary complex with a functional adaptability exploited in partner recognition and unravels the molecular determinants of the reported inhibitory action of CyaY in the IscS-IscU-CyaY complex explained in terms of the hampering effect on specific IscU-IscS movements. General significance Our study provides the first mechanistic basis to explain how the IscS-IscU complex selects its binding partners and supports the inhibitory role of CyaY in the ternary complex.

Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel.

Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested.

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels

Ferritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe2+-loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe2+ uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe2+ through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H′ protein and some of its variants as case studies. We describe the structural features that determine Fe2+ translocation with atomistic detail, and we propose a putative mechanism for Fe2+ transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin.

Smyd3 open & closed lock mechanism for substrate recruitment: The hinge motion of C-terminal domain inferred from μ-second molecular dynamics simulations

BACKGROUND The human lysine methyltransferase Smyd3, a member of the SET and MYND domain containing protein family, harbors methylation activity on both histone and non-histone targets in a tightly regulated manner. The mechanism of how Smyd3 dynamically regulates substrate recognition is still not fully unveiled. METHODS Here, we employed molecular dynamics simulations on full length human Smyd3, performed to a total of 1.2 μ-second, in the presence (holo) and absence (apo) of the S-Adenosyl methionine (AdoMet) cofactor. The dynamical features of Smyd3 in apo and holo states have been examined and compared via examining geometrical and electrostatic properties. RESULTS The results show a distinct dynamics of the C-terminal domain (CTD) in the two states. In the apo state, the CTD undergoes a large hinge like motion and samples more opened configurations, thus acting like a loosened clamp and resulting in expanded substrate binding crevice. In the holo state, the CTD exhibits a restricted motion while the overall structure remains compact, mimicking a closed clamp. This leads to a localized increase in the negative potential at the substrate binding cleft. Further, solvent accessibility of critical residues at the target lysine access channel, important for methylation activity, is increased. CONCLUSIONS We postulate that AdoMet cofactor acts like a key and locks Smyd3 in a closed conformation. In effect, the cofactor binding restricts the elasticity of the CTD, presenting a compact substrate binding cleft with high negative potential, which may have implications on substrate recruitment via long range electrostatics. GENERAL SIGNIFICANCE The deletion of the CTD from Smyd3 has been shown to abolish the basal histone methylation activity. Our study highlights the importance of the CTD elasticity in shaping the substrate binding site for recognition and supports the previously proposed role of the CTD in stabilizing the active site for methylation activity.

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.

Boundary condition effects on the dynamic and electric properties of hydration layers.

Water solvation has a central role in several biochemical processes ranging from protein folding to biomolecular recognition and enzyme catalysis. Because of its importance, the structure and dynamics of hydration layers around biological macromolecules have been the targets of a great number of experimental and computational studies. In the present contribution, we have investigated the effects of periodic boundary conditions (PBCs), as used in conjunction with molecular dynamics (MD) simulations, on the dynamic and electric properties of water layers. In particular, we have systematically performed MD simulations of neat water and biomolecules in aqueous solutions by imposing a different external dielectric constant, a generally overlooked parameter in PBC simulations. The effect of the system size has also been addressed. Overall, our results consistently indicate that the dipole moment properties of water layers, and specifically the dipole moment fluctuations and the reorientational correlation functions, can be sensitive to the choice of the external boundary conditions, whereas other molecular properties, such as the self-diffusion coefficient and the reorientational relaxation times, are not affected. We think that our investigation may help to assess appropriate simulation conditions for modeling the aqueous environment of relevant biochemical systems and processes.

Conformational Dynamics of Lysine Methyltransferase Smyd2. Insights into the Different Substrate Crevice Characteristics of Smyd2 and Smyd3

Smyd2, the SET and MYND domain containing protein lysine methyltransferase, targets histone and nonhistone substrates. Methylation of nonhistone substrates has direct implications in cancer development and progression. Dynamic regulation of Smyd2 activity and the structural basis of broad substrate specificity still remain elusive. Herein, we report on extensive molecular dynamics simulations on a full length Smyd2 in the presence and absence of AdoMet cofactor (covering together 1.3 μs of sampling), and the accompanying conformational transitions. Additionally, dynamics of the C-terminal domain (CTD) and structural features of substrate crevices of Smyd2 and Smyd3 are compared. The CTD of Smyd2 exhibits conformational flexibility in both states. In the holo form, however, it undergoes larger hinge motions resulting in more opened configurations than the apo form, which is confined around the partially open starting X-ray configuration. AdoMet binding triggers increased elasticity of the CTD leading Smyd2 to adopt fully opened configurations, which completely exposes the substrate binding crevice. These long-range concerted motions highlight Smyd2s ability to target substrates of varying sizes. Substrate crevices of Smyd2 and Smyd3 show distinct features in terms of spatial, hydration, and electrostatic properties that emphasize their characteristic modes of substrates interaction and entry pathways for inhibitor binding. On the whole, our study shows how the elasticity and hinge motion of the CTD regulate its functional role and underpin the basis of broad substrate specificity of Smyd2. We also highlight the specific structural principles that guide substrate and inhibitor binding to Smyd2 and Smyd3.