SummaryThe exchange of metabolites between the mitochondrial matrix and the cytosol depends on β-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and β-barrel protein biogenesis. Our work reveals how a single mitochondrial “transfer-chaperone” system is able to guide α-helical and β-barrel membrane proteins in a “nascent chain-like” conformation through a ribosome-free compartment.
Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg 2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition.
Determining the structures, kinetics, thermodynamics and mechanisms that underlie conformational exchange processes in proteins remains extremely difficult. Only in favourable cases is it possible to provide atomic-level descriptions of sparsely populated and transiently formed alternative conformations. Here we benchmark the ability of enhanced-sampling molecular dynamics simulations to determine the free energy landscape of the L99A cavity mutant of T4 lysozyme. We find that the simulations capture key properties previously measured by NMR relaxation dispersion methods including the structure of a minor conformation, the kinetics and thermodynamics of conformational exchange, and the effect of mutations. We discover a new tunnel that involves the transient exposure towards the solvent of an internal cavity, and show it to be relevant for ligand escape. Together, our results provide a comprehensive view of the structural landscape of a protein, and point forward to studies of conformational exchange in systems that are less characterized experimentally.DOI: http://dx.doi.org/10.7554/eLife.17505.001
The ability to predict accurate thermodynamic and kinetic properties in biomolecular systems is of both scientific and practical utility. While both remain very difficult, predictions of kinetics are particularly difficult because rates, in contrast to free energies, depend on the route taken. For this reason, specific enhanced sampling methods are needed to calculate long-time scale kinetics. It has recently been demonstrated that it is possible to recover kinetics through the so-called "infrequent metadynamics" simulations, where the simulations are biased in a way that minimally corrupts the dynamics of moving between metastable states. This method, however, requires the bias to be added slowly, thus hampering applications to processes with only modest separations of time scales. Here we present a frequency-adaptive strategy which bridges normal and infrequent metadynamics. We show that this strategy can improve the precision and accuracy of rate calculations at fixed computational cost and should be able to extend rate calculations for much slower kinetic processes.
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na + -driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na + - and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na + - and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
Biomolecular systems such as protein–ligand complexes are governed by thermodynamic and kinetic properties that may be estimated at the same time through enhanced-sampling molecular simulations.
Intrinsically disordered proteins (IDPs) exert their functions by binding to partner proteins via a complex process that includes coupled folding and binding. Because inhibiting the binding of the IDP p53 to its partner MDM2 has become a promising strategy for the design of anticancer drugs, we carried out metadynamics simulations to study the coupled folding and binding process linking the IDP p53 to MDM2 in atomic detail. Using bias-exchange metadynamics (BE-MetaD) and infrequent metadynamics (InMetaD), we estimated the binding free energy, the unbinding rate, and the binding rate. By analyzing the stable intermediates, we uncovered the role non-native interactions played in the p53-MDM2 binding/unbinding process. We used a three-state model to describe the whole binding/unbinding process and to obtain the corresponding rate constants. Our work shows that the binding of p53 favors an induced-fit mechanism which proceeds in a stepwise fashion. Our results can be helpful for gaining an in-depth understanding of the coupled folding and binding process needed for the design of MDM2 inhibitors.
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