142 research outputs found
Accessing a Hidden Conformation of the Maltose Binding Protein Using Accelerated Molecular Dynamics
Periplasmic binding proteins (PBPs) are a large family of molecular transporters that play a key role in nutrient uptake and chemotaxis in Gram-negative bacteria. All PBPs have characteristic two-domain architecture with a central interdomain ligand-binding cleft. Upon binding to their respective ligands, PBPs undergo a large conformational change that effectively closes the binding cleft. This conformational change is traditionally viewed as a ligand induced-fit process; however, the intrinsic dynamics of the protein may also be crucial for ligand recognition. Recent NMR paramagnetic relaxation enhancement (PRE) experiments have shown that the maltose binding protein (MBP) - a prototypical member of the PBP superfamily - exists in a rapidly exchanging (ns to Β΅s regime) mixture comprising an open state (approx 95%), and a minor partially closed state (approx 5%). Here we describe accelerated MD simulations that provide a detailed picture of the transition between the open and partially closed states, and confirm the existence of a dynamical equilibrium between these two states in apo MBP. We find that a flexible part of the protein called the balancing interface motif (residues 175β184) is displaced during the transformation. Continuum electrostatic calculations indicate that the repacking of non-polar residues near the hinge region plays an important role in driving the conformational change. Oscillations between open and partially closed states create variations in the shape and size of the binding site. The study provides a detailed description of the conformational space available to ligand-free MBP, and has implications for understanding ligand recognition and allostery in related proteins
SIMS: A Hybrid Method for Rapid Conformational Analysis
Proteins are at the root of many biological functions, often performing complex tasks as the result of large changes in their
structure. Describing the exact details of these conformational changes, however, remains a central challenge for
computational biology due the enormous computational requirements of the problem. This has engendered the
development of a rich variety of useful methods designed to answer specific questions at different levels of spatial,
temporal, and energetic resolution. These methods fall largely into two classes: physically accurate, but computationally
demanding methods and fast, approximate methods. We introduce here a new hybrid modeling tool, the Structured
Intuitive Move Selector (SIMS), designed to bridge the divide between these two classes, while allowing the benefits of both
to be seamlessly integrated into a single framework. This is achieved by applying a modern motion planning algorithm,
borrowed from the field of robotics, in tandem with a well-established protein modeling library. SIMS can combine precise
energy calculations with approximate or specialized conformational sampling routines to produce rapid, yet accurate,
analysis of the large-scale conformational variability of protein systems. Several key advancements are shown, including the
abstract use of generically defined moves (conformational sampling methods) and an expansive probabilistic
conformational exploration. We present three example problems that SIMS is applied to and demonstrate a rapid solution
for each. These include the automatic determination of οΎοΎactiveοΎοΎ residues for the hinge-based system Cyanovirin-N,
exploring conformational changes involving long-range coordinated motion between non-sequential residues in Ribose-
Binding Protein, and the rapid discovery of a transient conformational state of Maltose-Binding Protein, previously only
determined by Molecular Dynamics. For all cases we provide energetic validations using well-established energy fields,
demonstrating this framework as a fast and accurate tool for the analysis of a wide range of protein flexibility problems
Exploration of Multi-State Conformational Dynamics and Underlying Global Functional Landscape of Maltose Binding Protein
An increasing number of biological machines have been revealed to have more than two macroscopic states. Quantifying the underlying multiple-basin functional landscape is essential for understanding their functions. However, the present models seem to be insufficient to describe such multiple-state systems. To meet this challenge, we have developed a coarse grained triple-basin structure-based model with implicit ligand. Based on our model, the constructed functional landscape is sufficiently sampled by the brute-force molecular dynamics simulation. We explored maltose-binding protein (MBP) which undergoes large-scale domain motion between open, apo-closed (partially closed) and holo-closed (fully closed) states responding to ligand binding. We revealed an underlying mechanism whereby major induced fit and minor population shift pathways co-exist by quantitative flux analysis. We found that the hinge regions play an important role in the functional dynamics as well as that increases in its flexibility promote population shifts. This finding provides a theoretical explanation of the mechanistic discrepancies in PBP protein family. We also found a functional βbacktrackingβ behavior that favors conformational change. We further explored the underlying folding landscape in response to ligand binding. Consistent with earlier experimental findings, the presence of ligand increases the cooperativity and stability of MBP. This work provides the first study to explore the folding dynamics and functional dynamics under the same theoretical framework using our triple-basin functional model
ATPase Regulation in the Maltose Transporter
This thesis investigates the mechanism of activity-coupling in the maltose transporter of Escherichia coli (MalFGK2); the way ATP hydrolysis is prevented in the absence of maltose, and then enabled to drive maltose transport. Like other ATP binding cassette importers, MalFGK2 requires substrate to be presented by a peripheral substrate-binding protein, in this case the maltose binding protein (MBP). MBP predominantly adopts an βopenβ resting state, but undergoes a rotation of its two domains to a βclosedβ state after maltose binding. In the closed state MBP is able to activate MalFGK2 to stimulate ATP hydrolysis and maltose transport.
Engineered mutants of MBP have been used to test the responsiveness of the transporter to changes in the conformational state of its binding protein. Careful analysis of ATPase stimulus by wild type MBP indicates that the open state of MBP is able to bind the transporter and promote ATP hydrolysis in the absence of maltose. Another mutant MBP, able to adopt the closed state in the presence of either sucrose or maltose, demonstrates that ATP hydrolysis is activated by interactions between the transporter and binding protein, and not between the transporter and substrate. Further, using MBP mutants incorporating introduced disulfide bonds and inter-domain cross-linkers we show that the closed form of MBP is able to activate substantial, but not maximal, ATPase activity without adopting the open conformation or releasing its bound substrate.
It has been determined that both stable forms of MBP, maltose-bound-closed and unliganded-open, separately stimulate ATP hydrolysis from the transporter. In contrast, the substrate itself does not directly stimulate activity but instead activates hydrolysis by stabilizing the closed state of MBP. Taken together our data indicate that the transporter exists in equilibrium between an inactive resting state and conformations receptive to binding by the closed and open forms of MBP. This suggests that uncoupled ATP hydrolysis is prevented by destabilizing ATPase relevant conformations of the transporter, and that MBP activates hydrolysis by binding these conformations in its closed and open states
Impact of Ser17 phosphorylation on the conformational dynamics of the oncoprotein MDM2
MDM2 is an important oncoprotein
that downregulates the activity
of the tumor suppressor protein p53 via binding of its N-terminal
domain to the p53 transactivation domain. The first 24 residues of
the MDM2 N-terminal domain form an intrinsically disordered βlidβ
region that interconverts on a millisecond time scale between βopenβ
and βclosedβ states in unliganded MDM2. While the former
conformational state is expected to facilitate p53 binding, the latter
competes in a pseudo-substrate manner with p53 for its binding site.
Phosphorylation of serine 17 in the MDM2 lid region is thought to
modulate the equilibrium between βopenβ and βclosedβ
lid states, but contradictory findings on the favored lid conformational
state upon phosphorylation have been reported. Here, the nature of
the conformational states of MDM2 pSer17 and Ser17Asp variants was
addressed by means of enhanced sampling molecular dynamics simulations.
Detailed analyses of the computed lid conformational ensembles indicate
that both lid variants stabilize a βclosedβ state, with
respect to wild type. Nevertheless, the nature of the closed-state
conformational ensembles differs significantly between the pSer17
and Ser17Asp variants. Thus, care should be applied in the interpretation
of biochemical experiments that use phosphomimetic variants to model
the effects of phosphorylation on the structure and dynamics of this
disordered protein region
Probing structure, function and dynamics in bacterial primary and secondary transporter-associated binding proteins
Substrate binding proteins (SBPs) are ubiquitous in all life forms and have evolved to perform diverse physiological functions, such as in membrane transport, gene regulation, neurotransmission, and quorum sensing. It is quite astounding to observe such functional diversity among the SBPs even when they are restricted by their fold space. Therefore, the SBPs are an excellent set of proteins that can reveal how proteins evolution novel function in a structurally conserved/constrained fold. This study attempts to understand the phenomenon of affinity and specificity evolution in SBPs by combining a set of biochemical, biophysical, and structural studies on the SBPs involved in translocation of substrates across the membrane using ATP-binding cassette (ABC) transporters and tripartite ATP-independent periplasmic (TRAP) transporters in gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa, respectively. Additionally, experimental, and computational methods were used in conjunction to highlight the variation in the dynamics of these SBPs. The results from this study highlight an intricate role of dynamics in complementing the structural alterations that are required for high-affinity ligand binding. Moreover, first ever neutron structure of a SBP was determined during my study to delineate the extensive network of water in the binding cavities of the SBPs that help stabilize larger substrates by forming water-mediated hydrogen bond interactions with the bound substrates. Furthermore, structures of two SBPs from T. maritima were determined in both substrate-free (apo) and substrate-bound (holo) forms and subsequently used for computational molecular dynamics simulation to determine the variation in dynamics due to substrate-binding. The novel TRAP SBP identified in P. aeruginosa was identified as a promiscuous binder of several tricarboxylic acid cycle (TCA) cycle intermediates. A total of six SBP structures were determined using X-ray crystallography and one SBP structure was determined using neutron crystallography. Finally, experimental neutron scattering was used to experimentally characterize the picosecond to nanosecond dynamics in SBPs and highlighted differences in the translational, rotational, and internal dynamical signatures of two SBP isoforms. Overall, the findings of this study can be broadly applied in biotechnology and biosensor development by artificially engineer affinity or specificity for a particular ligand
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