Atomistic picture of conformational exchange in a T4 lysozyme cavity mutant: an experiment-guided molecular dynamics study

Vallurupalli, Pramodh; Chakrabarti, Nilmadhab; Pomès, Régis; Kay, Lewis E.

doi:10.1039/c5sc03886c

Cited by 35 publications

(35 citation statements)

References 103 publications

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“…4 b). The fluctuation in the F114 c 1 angle was seen in high-temperature simulations of the ES-3mut (15), suggesting that these two states are similar in energy (see Verifying the Simulated Excited State by Comparison with Experiment). Despite the F114 c 1 and cavity-volume differences between the IS and the ES-Anton, the back-calculated chemical shifts from these structures are not distinct and are equal to chemical shifts determined experimentally (Fig.…”

Section: Defining the Metastates And Intermediate State In The Anton mentioning

confidence: 93%

“…1). The internal distance between F114 and V103 was chosen as a metric here because V103 neighbors the buried L99A cavity, and has the largest chemical shift difference (D6) between the ground and the excited state in both 15 N and 13 C a spectra. In the following paragraphs, we describe the conformational differences that define each of these states as unique from the ground and excited states and show that these differences are manifested in the back-calculated chemical shifts, calculated using the SHIFTXþ algorithm (49).…”

Section: Defining the Metastates And Intermediate State In The Anton mentioning

confidence: 99%

“…L99A is a model system for studying ligand binding to buried protein cavities and protein excited states, and the conformational changes that govern these phenomena have been enigmatic despite nearly 25 years of experimental study (5,6,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Experimental studies of the L99A mutant of T4 lysozyme have focused on defining the structures of and transition times between the ground state, the excited state, and ligand-bound states.…”

Section: Introductionmentioning

confidence: 99%

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Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

Schiffer

Feher

Malmstrom

et al. 2016

Biophysical Journal

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Proteins commonly sample a number of conformational states to carry out their biological function, often requiring transitions from the ground state to higher-energy states. Characterizing the mechanisms that guide these transitions at the atomic level promises to impact our understanding of functional protein dynamics and energy landscapes. The leucine-99-toalanine (L99A) mutant of T4 lysozyme is a model system that has an experimentally well characterized excited sparsely populated state as well as a ground state. Despite the exhaustive study of L99A protein dynamics, the conformational changes that permit transitioning to the experimentally detected excited state (~3%, DG~2 kcal/mol) remain unclear. Here, we describe the transitions from the ground state to this sparsely populated excited state of L99A as observed through a single molecular dynamics (MD) trajectory on the Anton supercomputer. Aside from detailing the ground-to-excited-state transition, the trajectory samples multiple metastates and an intermediate state en route to the excited state. Dynamic motions between these states enable cavity surface openings large enough to admit benzene on timescales congruent with known rates for benzene binding. Thus, these fluctuations between rare protein states provide an atomic description of the concerted motions that illuminate potential path(s) for ligand binding. These results reveal, to our knowledge, a new level of complexity in the dynamics of buried cavities and their role in creating mobile defects that affect protein dynamics and ligand binding.

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Section: Defining the Metastates And Intermediate State In The Anton mentioning

confidence: 93%

Section: Defining the Metastates And Intermediate State In The Anton mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

Schiffer

Feher

Malmstrom

et al. 2016

Biophysical Journal

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“…However, the structure of this rare state shows that the cavity is occluded by the aromatic side-chain of Phe114 that prevents the binding of benzene so that the dynamic process that has been characterized is not relevant to ligand binding [11,12]. Oxygen and xenon binding studies have also been performed that have helped to identify hydrophobic cavities in the protein [13][14][15].…”

Section: Figmentioning

confidence: 99%

Atomic Resolution Mechanism of Ligand Binding to a Solvent Inaccessible Cavity in T4 Lysozyme

Mondal

Ahalawat

Pandit

et al. 2018

Preprint

Self Cite

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Ligand binding sites in proteins are often localized to deeply buried cavities, inaccessible to bulk solvent.Yet, in many cases binding of cognate ligands occurs rapidly. An intriguing system is presented by the L99A cavity mutant of T4 Lysozyme (L99A T4L) that rapidly binds benzene (~10 6 M -1 s -1 ). Although the protein has long served as a model system for protein thermodynamics and crystal structures of both free and benzene-bound L99A T4L are available, the kinetic pathways by which benzene reaches its solventinaccessible binding cavity remain elusive. The current work, using extensive molecular dynamics simulation, achieves this by capturing the complete process of spontaneous recognition of benzene by L99A T4L at atomistic resolution. A series of multi-microsecond unbiased molecular dynamics simulation trajectories unequivocally reveal how benzene, starting in bulk solvent, diffuses to the protein and spontaneously reaches the solvent inaccessible cavity of L99A T4L. The simulated and high-resolution Xray derived bound structures are in excellent agreement. A robust four-state Markov model, developed using cumulative 60 µs trajectories, identifies and quantifies multiple ligand binding pathways with low activation barriers. Interestingly, none of these identified binding pathways required large conformational changes for ligand access to the buried cavity. Rather, these involve transient but crucial opening of a channel to the cavity via subtle displacements in the positions of key helices (helix4/helix6, helix7/helix9) leading to rapid binding. Free energy simulations further elucidate that these channel-opening events would have been unfavorable in otherwise ligand-inactive wild type T4L. Taken together, by integrating experiments, these simulations provide unprecedented mechanistic insights into complete ligand recognition process in a buried cavity. By illustrating the power of subtle helix movements in opening up multiple pathways for ligand access, this work offers an alternate view of ligand recognition mechanism in a solvent-inaccessible cavity, contrary to common perception of single dominant pathway for ligand binding.

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“…Solution-state NMR has proven unique in its ability to detect and structurally characterise these functionally relevant states, whose lifetimes can be on the order of a few milliseconds and are otherwise 'invisible' to experimental measures [6][7][8] . Such states can play important roles in processes as diverse as protein folding, molecular recognition and catalysis, but remain challenging to characterise [9][10][11][12][13][14][15][16] .…”

Section: Introductionmentioning

confidence: 99%

Dynamic design: manipulation of millisecond timescale motions on the energy landscape of Cyclophilin A

Juárez-Jiménez

Gupta

Karunanithy

et al. 2018

Preprint

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Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they inter-convert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov State modelling (MSM) to explore these 'excited' conformational states . Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1ρ solution state NMR measurements. By accurately and reliably exploring functionally relevant, but sparsely populated conformations with milli-second lifetimes in silico, our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery.

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Atomistic picture of conformational exchange in a T4 lysozyme cavity mutant: an experiment-guided molecular dynamics study

Abstract: Relaxation-dispersion NMR techniques and molecular dynamics simulations have been used to understand how a cavity mutant of T4 lysozyme interconverts between two compact conformations.

Cited by 35 publications

References 103 publications

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

Capturing Invisible Motions in the Transition from Ground to Rare Excited States of T4 Lysozyme L99A

Atomic Resolution Mechanism of Ligand Binding to a Solvent Inaccessible Cavity in T4 Lysozyme

Dynamic design: manipulation of millisecond timescale motions on the energy landscape of Cyclophilin A

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