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In the multi-domain protein adenylate kinase, domain insertion facilitates cooperative folding while accommodating function at domain interfaces.

Giri Rao VV, Gosavi S - PLoS Comput. Biol. (2014)

Bottom Line: Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity.In AKE, these interactions help promote conformational dynamics limited catalysis.Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.

ABSTRACT
Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.

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Folding of AKE mutants with deleted inter-domain interfaces.All free energies are scaled by their respective kBTfs. N and U denote the native and the unfolded ensembles. The error bars represent twice the square root of the variance in the folding free energy and were calculated using a jackknife algorithm. The blue Xs mark the position of the deleted interfaces. (A) Cartoon of the folded state of ΔCORE-NMPi at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). (C) Q* and Q*NMP are strength-scaled fraction of native contacts, e.g., a contact with a strength of 1.2 is counted as 1.2, when formed, in the calculation of Q* and Q*NMP. The 2DFES plot with RCs of Q*NMP and Q* shows that NMP folds as in WT. (D) Cartoon of the folded state of ΔCORE-LIDi at Tf. (E) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The U ensemble shifts to higher Q*. (F) The 2DFES plot with RCs of Q*LID and Q* shows that the U ensemble has partially folded LID.
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pcbi-1003938-g005: Folding of AKE mutants with deleted inter-domain interfaces.All free energies are scaled by their respective kBTfs. N and U denote the native and the unfolded ensembles. The error bars represent twice the square root of the variance in the folding free energy and were calculated using a jackknife algorithm. The blue Xs mark the position of the deleted interfaces. (A) Cartoon of the folded state of ΔCORE-NMPi at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). (C) Q* and Q*NMP are strength-scaled fraction of native contacts, e.g., a contact with a strength of 1.2 is counted as 1.2, when formed, in the calculation of Q* and Q*NMP. The 2DFES plot with RCs of Q*NMP and Q* shows that NMP folds as in WT. (D) Cartoon of the folded state of ΔCORE-LIDi at Tf. (E) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The U ensemble shifts to higher Q*. (F) The 2DFES plot with RCs of Q*LID and Q* shows that the U ensemble has partially folded LID.

Mentions: AKE has two inter-domain interfaces (Fig. 2A, 2C). We computationally create two mutants: one with no CORE-NMP inter-domain interactions (ΔCORE-NMPi; Fig. 5A) and another with no CORE-LID interactions (ΔCORE-LIDi; Fig. 5D). Similar mutants have been experimentally created to study AKE function [22]. However, when some contacts of an interface residue are deleted in the C-α SBM, it is enthalpically less stable when folded. In order to preserve WT-like energetic stabilization for every residue at the interface, we appropriately scale the strength of the other contacts of that residue when creating both ΔCORE-NMPi and ΔCORE-LIDi. This is similar to converting an outward facing residue which contributes to inter-domain interactions into an inward facing one which contributes to intra-domain interactions. We simulate C-α SBMs of these mutants to understand the effect of inter-domain interactions on the folding cooperativity of AKE.


In the multi-domain protein adenylate kinase, domain insertion facilitates cooperative folding while accommodating function at domain interfaces.

Giri Rao VV, Gosavi S - PLoS Comput. Biol. (2014)

Folding of AKE mutants with deleted inter-domain interfaces.All free energies are scaled by their respective kBTfs. N and U denote the native and the unfolded ensembles. The error bars represent twice the square root of the variance in the folding free energy and were calculated using a jackknife algorithm. The blue Xs mark the position of the deleted interfaces. (A) Cartoon of the folded state of ΔCORE-NMPi at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). (C) Q* and Q*NMP are strength-scaled fraction of native contacts, e.g., a contact with a strength of 1.2 is counted as 1.2, when formed, in the calculation of Q* and Q*NMP. The 2DFES plot with RCs of Q*NMP and Q* shows that NMP folds as in WT. (D) Cartoon of the folded state of ΔCORE-LIDi at Tf. (E) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The U ensemble shifts to higher Q*. (F) The 2DFES plot with RCs of Q*LID and Q* shows that the U ensemble has partially folded LID.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230728&req=5

pcbi-1003938-g005: Folding of AKE mutants with deleted inter-domain interfaces.All free energies are scaled by their respective kBTfs. N and U denote the native and the unfolded ensembles. The error bars represent twice the square root of the variance in the folding free energy and were calculated using a jackknife algorithm. The blue Xs mark the position of the deleted interfaces. (A) Cartoon of the folded state of ΔCORE-NMPi at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). (C) Q* and Q*NMP are strength-scaled fraction of native contacts, e.g., a contact with a strength of 1.2 is counted as 1.2, when formed, in the calculation of Q* and Q*NMP. The 2DFES plot with RCs of Q*NMP and Q* shows that NMP folds as in WT. (D) Cartoon of the folded state of ΔCORE-LIDi at Tf. (E) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The U ensemble shifts to higher Q*. (F) The 2DFES plot with RCs of Q*LID and Q* shows that the U ensemble has partially folded LID.
Mentions: AKE has two inter-domain interfaces (Fig. 2A, 2C). We computationally create two mutants: one with no CORE-NMP inter-domain interactions (ΔCORE-NMPi; Fig. 5A) and another with no CORE-LID interactions (ΔCORE-LIDi; Fig. 5D). Similar mutants have been experimentally created to study AKE function [22]. However, when some contacts of an interface residue are deleted in the C-α SBM, it is enthalpically less stable when folded. In order to preserve WT-like energetic stabilization for every residue at the interface, we appropriately scale the strength of the other contacts of that residue when creating both ΔCORE-NMPi and ΔCORE-LIDi. This is similar to converting an outward facing residue which contributes to inter-domain interactions into an inward facing one which contributes to intra-domain interactions. We simulate C-α SBMs of these mutants to understand the effect of inter-domain interactions on the folding cooperativity of AKE.

Bottom Line: Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity.In AKE, these interactions help promote conformational dynamics limited catalysis.Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.

ABSTRACT
Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.

Show MeSH
Related in: MedlinePlus