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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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Conformational transitions of AKE and its mutants.All the conformational transition simulations are performed below Tf and the free energies are scaled by the simulation temperature (kBT). O is the open state and C is the closed state. The RCs used to obtain the conformational transition 2DFES are the distance between the centre of masses of CORE and LID residues (the CORE-LID dist) and the distance between the centre of masses of CORE and NMP (termed the CORE-NMP dist). (A) The conformational transition 2DFES for WT AKE shows that LID closes before NMP. (B) The ΔCORE-NMPi 2DFES shows that conformational transitions proceed mainly through a LID-open-NMP-closed state unlike WT. (C) In ΔCORE-LIDi, the mechanism of the transitions remains the same as in WT. However, the open state is very sparsely populated. (D) In CP-NMPcut and (E) in CP-LIDcut, the conformational transitions show a mechanism similar to that in WT AKE with most transitions occurring via the LID-closed-NMP-open ensemble.
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pcbi-1003938-g007: Conformational transitions of AKE and its mutants.All the conformational transition simulations are performed below Tf and the free energies are scaled by the simulation temperature (kBT). O is the open state and C is the closed state. The RCs used to obtain the conformational transition 2DFES are the distance between the centre of masses of CORE and LID residues (the CORE-LID dist) and the distance between the centre of masses of CORE and NMP (termed the CORE-NMP dist). (A) The conformational transition 2DFES for WT AKE shows that LID closes before NMP. (B) The ΔCORE-NMPi 2DFES shows that conformational transitions proceed mainly through a LID-open-NMP-closed state unlike WT. (C) In ΔCORE-LIDi, the mechanism of the transitions remains the same as in WT. However, the open state is very sparsely populated. (D) In CP-NMPcut and (E) in CP-LIDcut, the conformational transitions show a mechanism similar to that in WT AKE with most transitions occurring via the LID-closed-NMP-open ensemble.

Mentions: We perform conformational transition simulations of AKE using a previously developed dual-SBM [18], [19] whose open state is the same structure as that used to simulate folding. The closed state is introduced into this C-α SBM through the addition of 39 contacts (Fig. 1C, red contacts) whose minimum energy (native contact) distances are calculated from the ligand-bound structure, 1AKE.pdb, chain A [18]. The strength of these closed state specific contacts is tuned to populate both the open and closed state ensembles in each of the proteins (Table S1). The molecular mechanism of the conformational transition of WT AKE has a LID-closed NMP-open intermediate and its relevance to experiment has already been shown [19]. Here, we reproduce the WT results (Fig. 7A) and use the same dual-SBM to understand how perturbations to either domain interfaces or chain connectivity affect the mechanism of conformational transitions. All conformational transition simulations are performed below Tf where the proteins do not unfold (See SI Methods and Table S1 for details). Also, at these temperatures the CPs of AKE are fully folded.


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)

Conformational transitions of AKE and its mutants.All the conformational transition simulations are performed below Tf and the free energies are scaled by the simulation temperature (kBT). O is the open state and C is the closed state. The RCs used to obtain the conformational transition 2DFES are the distance between the centre of masses of CORE and LID residues (the CORE-LID dist) and the distance between the centre of masses of CORE and NMP (termed the CORE-NMP dist). (A) The conformational transition 2DFES for WT AKE shows that LID closes before NMP. (B) The ΔCORE-NMPi 2DFES shows that conformational transitions proceed mainly through a LID-open-NMP-closed state unlike WT. (C) In ΔCORE-LIDi, the mechanism of the transitions remains the same as in WT. However, the open state is very sparsely populated. (D) In CP-NMPcut and (E) in CP-LIDcut, the conformational transitions show a mechanism similar to that in WT AKE with most transitions occurring via the LID-closed-NMP-open ensemble.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003938-g007: Conformational transitions of AKE and its mutants.All the conformational transition simulations are performed below Tf and the free energies are scaled by the simulation temperature (kBT). O is the open state and C is the closed state. The RCs used to obtain the conformational transition 2DFES are the distance between the centre of masses of CORE and LID residues (the CORE-LID dist) and the distance between the centre of masses of CORE and NMP (termed the CORE-NMP dist). (A) The conformational transition 2DFES for WT AKE shows that LID closes before NMP. (B) The ΔCORE-NMPi 2DFES shows that conformational transitions proceed mainly through a LID-open-NMP-closed state unlike WT. (C) In ΔCORE-LIDi, the mechanism of the transitions remains the same as in WT. However, the open state is very sparsely populated. (D) In CP-NMPcut and (E) in CP-LIDcut, the conformational transitions show a mechanism similar to that in WT AKE with most transitions occurring via the LID-closed-NMP-open ensemble.
Mentions: We perform conformational transition simulations of AKE using a previously developed dual-SBM [18], [19] whose open state is the same structure as that used to simulate folding. The closed state is introduced into this C-α SBM through the addition of 39 contacts (Fig. 1C, red contacts) whose minimum energy (native contact) distances are calculated from the ligand-bound structure, 1AKE.pdb, chain A [18]. The strength of these closed state specific contacts is tuned to populate both the open and closed state ensembles in each of the proteins (Table S1). The molecular mechanism of the conformational transition of WT AKE has a LID-closed NMP-open intermediate and its relevance to experiment has already been shown [19]. Here, we reproduce the WT results (Fig. 7A) and use the same dual-SBM to understand how perturbations to either domain interfaces or chain connectivity affect the mechanism of conformational transitions. All conformational transition simulations are performed below Tf where the proteins do not unfold (See SI Methods and Table S1 for details). Also, at these temperatures the CPs of AKE are fully folded.

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