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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 circular permutants.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. (A) Cartoon of the folded state of CP-NMPcut at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The N ensemble shifts to lower Q as compared to WT. (C) The 2DFES plot with RCs of QNMP and Q. NMP does not fold completely in the N ensemble. (D) Cartoon of the folded state of CP-LIDcut at Tf. (E) The FEP (black) shows that the barrier to folding is lower than that in WT AKE (grey). The N ensemble shifts to lower Q as compared to WT. (F) The 2DFES plot with RCs of QLID and Q shows a significantly higher population of unfolded LID in the N ensemble.
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pcbi-1003938-g006: Folding of AKE circular permutants.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. (A) Cartoon of the folded state of CP-NMPcut at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The N ensemble shifts to lower Q as compared to WT. (C) The 2DFES plot with RCs of QNMP and Q. NMP does not fold completely in the N ensemble. (D) Cartoon of the folded state of CP-LIDcut at Tf. (E) The FEP (black) shows that the barrier to folding is lower than that in WT AKE (grey). The N ensemble shifts to lower Q as compared to WT. (F) The 2DFES plot with RCs of QLID and Q shows a significantly higher population of unfolded LID in the N ensemble.

Mentions: We computationally generate CPs of AKE by connecting the WT termini (Fig. 2B). New N- and C-termini are created before NMP in CP-NMPcut and before LID in CP-LIDcut (Fig. 2B, C). This converts the inserted NMP and LID domains into singly-linked domains in the CPs (Fig. 6A, D). The C-α SBMs for both CPs have the same native contacts as WT AKE renumbered according to their changes in topology (Fig. 1C, 2C). Two clusters of hydrophobic residues, one in NMP (V39, A49 and M53) and the other in the flanking helices of LID (I116, V117, V164 and L168) stabilize the two inserted domains [31]. The cuts in both CPs are before these clusters and do not disturb their connectivity. The native contacts from these clusters have the same stabilizing effect on NMP and LID in the CPs as in WT. Thus, any changes in folding cooperativity are only due to the conversion of NMP or LID to singly-linked domains.


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 circular permutants.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. (A) Cartoon of the folded state of CP-NMPcut at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The N ensemble shifts to lower Q as compared to WT. (C) The 2DFES plot with RCs of QNMP and Q. NMP does not fold completely in the N ensemble. (D) Cartoon of the folded state of CP-LIDcut at Tf. (E) The FEP (black) shows that the barrier to folding is lower than that in WT AKE (grey). The N ensemble shifts to lower Q as compared to WT. (F) The 2DFES plot with RCs of QLID and Q shows a significantly higher population of unfolded LID in the N ensemble.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003938-g006: Folding of AKE circular permutants.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. (A) Cartoon of the folded state of CP-NMPcut at Tf. (B) The FEP (black with blue error bars) shows a free energy barrier similar to that in WT (grey). The N ensemble shifts to lower Q as compared to WT. (C) The 2DFES plot with RCs of QNMP and Q. NMP does not fold completely in the N ensemble. (D) Cartoon of the folded state of CP-LIDcut at Tf. (E) The FEP (black) shows that the barrier to folding is lower than that in WT AKE (grey). The N ensemble shifts to lower Q as compared to WT. (F) The 2DFES plot with RCs of QLID and Q shows a significantly higher population of unfolded LID in the N ensemble.
Mentions: We computationally generate CPs of AKE by connecting the WT termini (Fig. 2B). New N- and C-termini are created before NMP in CP-NMPcut and before LID in CP-LIDcut (Fig. 2B, C). This converts the inserted NMP and LID domains into singly-linked domains in the CPs (Fig. 6A, D). The C-α SBMs for both CPs have the same native contacts as WT AKE renumbered according to their changes in topology (Fig. 1C, 2C). Two clusters of hydrophobic residues, one in NMP (V39, A49 and M53) and the other in the flanking helices of LID (I116, V117, V164 and L168) stabilize the two inserted domains [31]. The cuts in both CPs are before these clusters and do not disturb their connectivity. The native contacts from these clusters have the same stabilizing effect on NMP and LID in the CPs as in WT. Thus, any changes in folding cooperativity are only due to the conversion of NMP or LID to singly-linked domains.

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