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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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Structures and contact maps of mutants of AKE.Domains of AKE are colored as in Fig. 1 (CORE: grey, NMP: green, LID: orange). (A) Interface deletion mutants of AKE. All marked residues are represented by their C-α atoms. The CORE-NMP interface is composed of interactions between the green (NMP) and the blue (CORE) residues. The mutant, ΔCORE-NMPi, has these interactions deleted. The CORE-LID interface is composed of interactions between the orange (LID) and the yellow (CORE) residues. This interface is deleted in the mutant ΔCORE-LIDi. (B) Circular permutants of AKE. The WT N- and C-termini are linked by a 4 glycine loop (dotted black line). New N- and C-termini are generated by cutting at one of the positions indicated by the circles (CP-NMPcut: green; between residues 29 and 30; CP-LIDcut: orange; between residues 111 and 112). (C) The C-α contact map of the open state of WT AKE with the intra-domain contacts colored as in Fig. 1C. The CORE-NMP interface interactions (33 blue contacts) are deleted in ΔCORE-NMPi while the CORE-LID interface interactions (22 yellow contacts) are deleted in ΔCORE-LIDi. The absolute contact order of the interfaces is: CORE-NMP interface: 29.58 and CORE-LID interface: 39.73. Colored circles (CP-NMPcut: green and CP-LIDcut: orange) mark the (x, x) location of the first residue x, of the CPs. The closed state specific contacts (Fig. 1C, red contacts; appropriately renumbered in the CPs) are not shown here but are present in the conformational transition simulations.
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pcbi-1003938-g002: Structures and contact maps of mutants of AKE.Domains of AKE are colored as in Fig. 1 (CORE: grey, NMP: green, LID: orange). (A) Interface deletion mutants of AKE. All marked residues are represented by their C-α atoms. The CORE-NMP interface is composed of interactions between the green (NMP) and the blue (CORE) residues. The mutant, ΔCORE-NMPi, has these interactions deleted. The CORE-LID interface is composed of interactions between the orange (LID) and the yellow (CORE) residues. This interface is deleted in the mutant ΔCORE-LIDi. (B) Circular permutants of AKE. The WT N- and C-termini are linked by a 4 glycine loop (dotted black line). New N- and C-termini are generated by cutting at one of the positions indicated by the circles (CP-NMPcut: green; between residues 29 and 30; CP-LIDcut: orange; between residues 111 and 112). (C) The C-α contact map of the open state of WT AKE with the intra-domain contacts colored as in Fig. 1C. The CORE-NMP interface interactions (33 blue contacts) are deleted in ΔCORE-NMPi while the CORE-LID interface interactions (22 yellow contacts) are deleted in ΔCORE-LIDi. The absolute contact order of the interfaces is: CORE-NMP interface: 29.58 and CORE-LID interface: 39.73. Colored circles (CP-NMPcut: green and CP-LIDcut: orange) mark the (x, x) location of the first residue x, of the CPs. The closed state specific contacts (Fig. 1C, red contacts; appropriately renumbered in the CPs) are not shown here but are present in the conformational transition simulations.

Mentions: Structure-based models (SBMs) capture the funnelled energy landscape of proteins [33] by encoding the native structure into their potential energy functions [34]. MD simulations of SBMs have successfully reproduced the folding routes and the folding rates of diverse proteins [1], [8], [9], [12], [32], [34]. We find in agreement with experiment that a C-α structure-based model (C-α SBM) of AKE folds cooperatively. In order to test the role of inter-domain interactions in this cooperative folding, we create AKE mutants where these interactions are deleted (Fig. 2A, 2C). MD simulations of these mutants show that the inter-domain interactions play a minimal role in promoting folding cooperativity. We then create circular permutants (CPs) of AKE where either LID or NMP (the inserted domains) are converted to singly-linked domains (Fig. 2B, 2C) and find that the CPs fold less cooperatively than WT AKE. Thus, domain insertion rather than inter-domain interactions promotes folding cooperativity in 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)

Structures and contact maps of mutants of AKE.Domains of AKE are colored as in Fig. 1 (CORE: grey, NMP: green, LID: orange). (A) Interface deletion mutants of AKE. All marked residues are represented by their C-α atoms. The CORE-NMP interface is composed of interactions between the green (NMP) and the blue (CORE) residues. The mutant, ΔCORE-NMPi, has these interactions deleted. The CORE-LID interface is composed of interactions between the orange (LID) and the yellow (CORE) residues. This interface is deleted in the mutant ΔCORE-LIDi. (B) Circular permutants of AKE. The WT N- and C-termini are linked by a 4 glycine loop (dotted black line). New N- and C-termini are generated by cutting at one of the positions indicated by the circles (CP-NMPcut: green; between residues 29 and 30; CP-LIDcut: orange; between residues 111 and 112). (C) The C-α contact map of the open state of WT AKE with the intra-domain contacts colored as in Fig. 1C. The CORE-NMP interface interactions (33 blue contacts) are deleted in ΔCORE-NMPi while the CORE-LID interface interactions (22 yellow contacts) are deleted in ΔCORE-LIDi. The absolute contact order of the interfaces is: CORE-NMP interface: 29.58 and CORE-LID interface: 39.73. Colored circles (CP-NMPcut: green and CP-LIDcut: orange) mark the (x, x) location of the first residue x, of the CPs. The closed state specific contacts (Fig. 1C, red contacts; appropriately renumbered in the CPs) are not shown here but are present in the conformational transition simulations.
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pcbi-1003938-g002: Structures and contact maps of mutants of AKE.Domains of AKE are colored as in Fig. 1 (CORE: grey, NMP: green, LID: orange). (A) Interface deletion mutants of AKE. All marked residues are represented by their C-α atoms. The CORE-NMP interface is composed of interactions between the green (NMP) and the blue (CORE) residues. The mutant, ΔCORE-NMPi, has these interactions deleted. The CORE-LID interface is composed of interactions between the orange (LID) and the yellow (CORE) residues. This interface is deleted in the mutant ΔCORE-LIDi. (B) Circular permutants of AKE. The WT N- and C-termini are linked by a 4 glycine loop (dotted black line). New N- and C-termini are generated by cutting at one of the positions indicated by the circles (CP-NMPcut: green; between residues 29 and 30; CP-LIDcut: orange; between residues 111 and 112). (C) The C-α contact map of the open state of WT AKE with the intra-domain contacts colored as in Fig. 1C. The CORE-NMP interface interactions (33 blue contacts) are deleted in ΔCORE-NMPi while the CORE-LID interface interactions (22 yellow contacts) are deleted in ΔCORE-LIDi. The absolute contact order of the interfaces is: CORE-NMP interface: 29.58 and CORE-LID interface: 39.73. Colored circles (CP-NMPcut: green and CP-LIDcut: orange) mark the (x, x) location of the first residue x, of the CPs. The closed state specific contacts (Fig. 1C, red contacts; appropriately renumbered in the CPs) are not shown here but are present in the conformational transition simulations.
Mentions: Structure-based models (SBMs) capture the funnelled energy landscape of proteins [33] by encoding the native structure into their potential energy functions [34]. MD simulations of SBMs have successfully reproduced the folding routes and the folding rates of diverse proteins [1], [8], [9], [12], [32], [34]. We find in agreement with experiment that a C-α structure-based model (C-α SBM) of AKE folds cooperatively. In order to test the role of inter-domain interactions in this cooperative folding, we create AKE mutants where these interactions are deleted (Fig. 2A, 2C). MD simulations of these mutants show that the inter-domain interactions play a minimal role in promoting folding cooperativity. We then create circular permutants (CPs) of AKE where either LID or NMP (the inserted domains) are converted to singly-linked domains (Fig. 2B, 2C) and find that the CPs fold less cooperatively than WT AKE. Thus, domain insertion rather than inter-domain interactions promotes folding cooperativity in 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