Dataset Information

N-terminal strands of filamin Ig domains act as a conformational switch under biological forces.

ABSTRACT: Conformational changes of filamin A under stress have been postulated to play crucial roles in signaling pathways of cell responses. Direct observation of conformational changes under stress is beyond the resolution of current experimental techniques. On the other hand, computational studies are mainly limited to either traditional molecular dynamics simulations of short durations and high forces or simulations of simplified models. Here we perform all-atom discrete molecular dynamics (DMD) simulations to study thermally and force-induced unfolding of filamin A. The high conformational sampling efficiency of DMD allows us to observe force-induced unfolding of filamin A Ig domains under physiological forces. The computationally identified critical unfolding forces agree well with experimental measurements. Despite a large heterogeneity in the population of force-induced intermediate states, we find a common initial unfolding intermediate in all the Ig domains of filamin, where the N-terminal strand unfolds. We also study the thermal unfolding of several filamin Ig-like domains. We find that thermally induced unfolding features an early-stage intermediate state similar to the one observed in force-induced unfolding and characterized by the N-terminal strand being unfurled. We propose that the N-terminal strand may act as a conformational switch that unfolds under physiological forces leading to exposure of cryptic binding sites, removal of native binding sites, and modulating the quaternary structure of domains.

SUBMITTER: Kesner BA

PROVIDER: S-EPMC2804786 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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N-terminal strands of filamin Ig domains act as a conformational switch under biological forces.

Kesner Barry A BA Ding Feng F Temple Brenda R BR Dokholyan Nikolay V NV

Proteins 20100101 1

Conformational changes of filamin A under stress have been postulated to play crucial roles in signaling pathways of cell responses. Direct observation of conformational changes under stress is beyond the resolution of current experimental techniques. On the other hand, computational studies are mainly limited to either traditional molecular dynamics simulations of short durations and high forces or simulations of simplified models. Here we perform all-atom discrete molecular dynamics (DMD) simu ...[more]

PMID: 19514078

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Project description:Introduction: Protein conformational switches are often constructed by fusing an input domain, which recognizes a target ligand, to an output domain that establishes a biological response. Prior designs have employed binding-induced folding of the input domain to drive a conformational change in the output domain. Adding a second input domain can in principle harvest additional binding energy for performing useful work. It is not obvious, however, how to fuse two binding domains to a single output domain such that folding of both binding domains combine to effect conformational change in the output domain. Methods: Here, we converted the ribonuclease barnase (Bn) to a switchable enzyme by duplicating a C-terminal portion of its sequence and appending it to its N-terminus, thereby establishing a native fold (OFF state) and a circularly permuted fold (ON state) that competed for the shared core in a mutually exclusive fashion. Two copies of FK506 binding protein (FKBP), both made unstable by the V24A mutation and one that had been circularly permuted, were inserted into the engineered barnase at the junctions between the shared and duplicated sequences. Results: Rapamycin-induced folding of FK506 binding protein stretched and unfolded the native fold of barnase via the mutually exclusive folding effect, and rapamycin-induced folding of permuted FK506 binding protein stabilized the permuted fold of barnase by the loop-closure entropy principle. These folding events complemented each other to turn on RNase function. The cytotoxic switching mechanism was validated in yeast and human cells, and in vitro with purified protein. Discussion: Thermodynamic modeling and experimental results revealed that the dual action of loop-closure entropy and mutually exclusive folding is analogous to an engine transmission in which loop-closure entropy acts as the low gear, providing efficient switching at low ligand concentrations, and mutually exclusive folding acts as the high gear to allow the switch to reach its maximum response at high ligand concentrations.

Dataset Information

N-terminal strands of filamin Ig domains act as a conformational switch under biological forces.

Publications

N-terminal strands of filamin Ig domains act as a conformational switch under biological forces.

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