The Open Nucleotide Pocket of the Profilin/Actin X-Ray Structure Is Unstable and Closes in the Absence of Profilin

Minehardt, Todd J.; Kollman, Peter A.; Cooke, Roger; Pate, Edward

doi:10.1529/biophysj.105.072900

Cited by 26 publications

(29 citation statements)

References 42 publications

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“…An open cleft is defined as an increased separation of Ϸ3 Å between two ␤-hairpins that are located on either side of the nucleotide (residues 11-16 in subdomain 1 and residues 154 -161 in subdomain 3) at the base of the nucleotide cleft. Recent molecular dynamic simulations show that the open nucleotide cleft of actin-profilin reverts to its preferred closed cleft conformation once the profilin is removed from the simulation (26). The authors conclude from this molecular dynamics study that there is no thermodynamically stable open cleft structure of monomeric actin.…”

Section: Discussionmentioning

confidence: 83%

Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States

Rould¹,

Wan²,

Joel³

et al. 2006

J. Biol. Chem.

129

170

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ATP hydrolysis drives actin filament dynamics. The effect known as treadmilling arises from the preferential addition of ATP-actin monomers to the plus or barbed end of actin and the preferential dissociation of ADP-actin from the minus or pointed end. Hydrolysis of ATP occurs after the monomer is incorporated into the filament. In addition, actin-binding proteins often have significantly different affinities for the ADPbound versus ATP-bound forms of actin. Both of these lines of evidence strongly suggest that there must be significant structural changes in actin induced by ATP hydrolysis. Indirect solution techniques such as proteolytic digestion rates and fluorescence studies (reviewed in Ref. 1) are in agreement with conformational differences between the ADP and ATP states, but direct structural evidence has been lacking until recently.Based on crystal structures of a tetramethylrhodaminelabeled monomeric actin (TMR-actin) 2 with ADP (2) or AMP-PNP (3) at the active site, Dominguez and co-workers proposed the provocative idea that ATP hydrolysis initiates a series of changes originating at the active site, that ultimately cause a loop-to-helix transition in the DNase binding loop in subdomain 2 of actin ("D-loop," residues in subdomain 2 of actin which comprise part of the DNase I binding site). They suggested that this was the long sought after change in structure between ADP and ATP actin. The cleft between subdomains 2 and 4 of actin remained closed in both nucleotide states. Despite this observation, an opposing point of view (4) held that the more important conformational change is an opening of the cleft upon ATP hydrolysis, a change that would be compatible with that observed for other nucleotide hydrolyzing proteins. Docking of actin crystal structures into negatively stained images of F-actin was also consistent with the idea that ADPactin had a more open cleft than the triphosphate state (5). If the latter view is correct, one would have to suppose that the modification of Cys 374 by TMR stabilized the closed conformation and inactivated the nucleotide sensing mechanism by virtue of the binding of rhodamine between subdomains 1 and 3, a potential hinge region (6) of the molecule. It has been suggested (4) that the short helix observed in the crystal structure of the ADP state of TMR-actin could be formed as a result of contacts with neighboring molecules in the crystal. We provide further evidence to support this idea, and suggest a pathway through which nucleotide-dependent changes may propagate through fortuitous crystal packing interactions from one monomer to the D-loop region of another in the TMR-actin crystals.Here we crystallized and expressed a cytoplasmic actin that was rendered incapable of polymerization by virtue of two surface mutations in subdomain 4 (A204E/P243K). This strategy negates concerns raised about the TMR modification of actin.De novo crystallization of AP-actin with either ATP or ADP at the active site reveals obligatory nucleotide-dependent confor-* This work was suppor...

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Section: Discussionmentioning

confidence: 83%

Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States

Rould¹,

Wan²,

Joel³

et al. 2006

J. Biol. Chem.

129

170

View full text Add to dashboard Cite

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“…8 MD simulations have shown that removal of profilin from the open actin-profilin complex results in the closure of the nucleotide binding cleft of actin on nanosecond timescales. 25 Three lines of evidence support the hypothesis that the C-terminus of Arp3 acts like an intrinsic profilin. First, among the nine currently available crystal structures of the Arp2/3 complex, the 5 C-terminal residues of Arp3 that extend beyond the C-terminus of actin show a greater tendency to be ordered when the nucleotide cleft of Arp3 is open than when it is closed.…”

Section: Structural Basis For Differences Between Actin and Arp3mentioning

confidence: 86%

“…21 Simulations of actin dynamics have run the gamut from density functional calculations of the active site to coarse-grained filamentous actin networks. [22][23][24][25][26][27][28][29] All-atom simulations on nanosecond timescales have been helpful in complementing structural experiments. Most relevant to this study are molecular dynamics (MD) simulations of monomeric actin up to 50 ns in explicit water, where the nucleotide binding cleft of actin stayed closed when bound to both ATP and ADP.…”

Section: Introductionmentioning

confidence: 99%

“…29 A simulation of actin-profilin showed that the open nucleotide binding cleft as observed in crystal structures 14 was unstable when profilin was replaced with water. 25 Here, we used MD simulations to study conformations of actin and Arp3 in a variety of nucleotide-bound states on the nanosecond timescale. These simulations have given us insights into several aspects of the effect of nucleotides on actin family proteins, including differences in the overall flexibility of actin and Arp3, responses of residues lining the nucleotide cleft, relay of structural changes outward from the cleft, pathways for partial release of the nucleotide, and the effect of the divalent cation on protein dynamics.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Nucleotide-Mediated Conformational Changes of Monomeric Actin and Arp3 Studied by Molecular Dynamics Simulations

Dalhaimer

Pollard

Nolen

2008

Journal of Molecular Biology

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Members of the actin family of proteins exhibit different biochemical properties when ATP, ADP-P i , ADP, or no nucleotide is bound. We used molecular dynamics simulations to study the effect of nucleotides on the behavior of actin and actin-related protein 3 (Arp3). In all of the actin simulations, the nucleotide cleft stayed closed, as in most crystal structures. ADP was much more mobile within the cleft than ATP, despite the fact that both nucleotides adopt identical conformations in actin crystal structures. The nucleotide cleft of Arp3 opened in most simulations with ATP, ADP, and no bound nucleotide. Deletion of a C-terminal region of Arp3 that extends beyond the conserved actin sequence reduced the tendency of the Arp3 cleft to open. When the Arp3 cleft opened, we observed multiple instances of partial release of the nucleotide. Cleft opening in Arp3 also allowed us to observe correlated movements of the phosphate clamp, cleft mouth, and barbed-end groove, providing a way for changes in the nucleotide state to be relayed to other parts of Arp3. The DNase binding loop of actin was highly flexible regardless of the nucleotide state. The conformation of Ser14/Thr14 in the P1 loop was sensitive to the presence of the γ-phosphate, but other changes observed in crystal structures were not correlated with the nucleotide state on nanosecond timescales. The divalent cation occupied three positions in the nucleotide cleft, one of which was not previously observed in actin or Arp2/3 complex structures. In sum, these simulations show that subtle differences in structures of actin family proteins have profound effects on their nucleotide-driven behavior.

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“…Some biochemical observations have also been interpreted as evidence of a more open cleft in actin than suggested by the majority of the crystal structures (37). However, a wide-open cleft appears to be structurally unstable (38). More important, the wide-open structure of profilin-␤-actin (36) was obtained in an unconventional way, by transferring the original crystals (20) into a high-phosphate solution.…”

Section: Structure Of Unphosphorylated Dictyostelium Actin Complexed mentioning

confidence: 99%

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

Baek

Xiong

Ferrón

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-␤-actin and profilin-␣-actin. By comparing this representative set of profilin-actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7°rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin-actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.actin phosphorylation ͉ profilin-actin structure ͉ pY53-actin structure ͉ Dictyostelium discoideum actin ͉ gelsolin-actin structure

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The Open Nucleotide Pocket of the Profilin/Actin X-Ray Structure Is Unstable and Closes in the Absence of Profilin

Cited by 26 publications

References 42 publications

Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States

Crystal Structures of Expressed Non-polymerizable Monomeric Actin in the ADP and ATP States

Nucleotide-Mediated Conformational Changes of Monomeric Actin and Arp3 Studied by Molecular Dynamics Simulations

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

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