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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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Contact clusters and free energy profiles of WT AKE.(A) The three contact clusters whose contacts form and break together during the folding and unfolding of WT AKE are projected onto their residues and shown on the structure: CORE-N (black; residues 1–29 and 68–117), LID (orange) and CORE-C (grey; residues 161–214). No contact cluster was found in NMP (green). (B) The contacts of the residues that form the clusters are shown in the same colors as in A. The intra-LID and intra-NMP contacts are enclosed in orange and green boxes and their contact location close to the diagonal underlines that LID and NMP are inserted domains. The CORE-N contacts are demarcated by the black box. (C–G) Reaction coordinates (RCs) for the folding free energy plots are defined as the fraction of native contacts formed in the whole protein (Q; all contacts, Fig. 2C), CORE-N (QCORE-N; black contacts in B), CORE-C (QCORE-C; grey contacts in B), CORE (QCORE; grey contacts, Fig. 2C), LID (QLID; orange contacts, Fig. 2C) and NMP (QNMP; green, Fig. 2C). The more folded a given region, the higher the value of the corresponding Q. (C) The scaled folding free energy (ΔG/kBTf) of WT AKE plotted as a function of Q. 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. (D) 2DFES plotted with RCs of QCORE and Q. The free energies are scaled by kBTf for all 2DFESs. The color indicates the height of the free energy at a given value of (x, y) and the limits of the color scale are the same as the limits of the y-axis in C. (E) The 2DFES plot with RCs of QLID and Q. (F) The 2DFES plot with RCs of QNMP and Q. (G) The 2DFES plot with RCs of QCORE-N and QCORE-C shows that CORE-N folds before CORE-C in the predominant folding route.
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pcbi-1003938-g003: Contact clusters and free energy profiles of WT AKE.(A) The three contact clusters whose contacts form and break together during the folding and unfolding of WT AKE are projected onto their residues and shown on the structure: CORE-N (black; residues 1–29 and 68–117), LID (orange) and CORE-C (grey; residues 161–214). No contact cluster was found in NMP (green). (B) The contacts of the residues that form the clusters are shown in the same colors as in A. The intra-LID and intra-NMP contacts are enclosed in orange and green boxes and their contact location close to the diagonal underlines that LID and NMP are inserted domains. The CORE-N contacts are demarcated by the black box. (C–G) Reaction coordinates (RCs) for the folding free energy plots are defined as the fraction of native contacts formed in the whole protein (Q; all contacts, Fig. 2C), CORE-N (QCORE-N; black contacts in B), CORE-C (QCORE-C; grey contacts in B), CORE (QCORE; grey contacts, Fig. 2C), LID (QLID; orange contacts, Fig. 2C) and NMP (QNMP; green, Fig. 2C). The more folded a given region, the higher the value of the corresponding Q. (C) The scaled folding free energy (ΔG/kBTf) of WT AKE plotted as a function of Q. 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. (D) 2DFES plotted with RCs of QCORE and Q. The free energies are scaled by kBTf for all 2DFESs. The color indicates the height of the free energy at a given value of (x, y) and the limits of the color scale are the same as the limits of the y-axis in C. (E) The 2DFES plot with RCs of QLID and Q. (F) The 2DFES plot with RCs of QNMP and Q. (G) The 2DFES plot with RCs of QCORE-N and QCORE-C shows that CORE-N folds before CORE-C in the predominant folding route.

Mentions: Both the conformational transitions and the folding of AKE have previously been studied using different flavors of SBMs [18], [19], [32]. Here, we use a well-tested SBM [34] which uses only the C-α atom to represent the entire residue. This C-α SBM, which uses the folded structure (Fig. 1A, 2A, 2B) and its contact map (Fig. 1C, 2C) as inputs, is sufficient to capture the main changes in folding upon topological perturbation [35], [36] and is not intended for the detailed analysis of structural populations [2], [32]. We first validate the C-α SBM by performing folding simulations of WT AKE (Fig. 3) and show that these broadly agree with results from diverse ensemble experiments (HX-NMR [31], tryptophan fluorescence [24], and time-resolved FRET [26]–[30]).


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)

Contact clusters and free energy profiles of WT AKE.(A) The three contact clusters whose contacts form and break together during the folding and unfolding of WT AKE are projected onto their residues and shown on the structure: CORE-N (black; residues 1–29 and 68–117), LID (orange) and CORE-C (grey; residues 161–214). No contact cluster was found in NMP (green). (B) The contacts of the residues that form the clusters are shown in the same colors as in A. The intra-LID and intra-NMP contacts are enclosed in orange and green boxes and their contact location close to the diagonal underlines that LID and NMP are inserted domains. The CORE-N contacts are demarcated by the black box. (C–G) Reaction coordinates (RCs) for the folding free energy plots are defined as the fraction of native contacts formed in the whole protein (Q; all contacts, Fig. 2C), CORE-N (QCORE-N; black contacts in B), CORE-C (QCORE-C; grey contacts in B), CORE (QCORE; grey contacts, Fig. 2C), LID (QLID; orange contacts, Fig. 2C) and NMP (QNMP; green, Fig. 2C). The more folded a given region, the higher the value of the corresponding Q. (C) The scaled folding free energy (ΔG/kBTf) of WT AKE plotted as a function of Q. 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. (D) 2DFES plotted with RCs of QCORE and Q. The free energies are scaled by kBTf for all 2DFESs. The color indicates the height of the free energy at a given value of (x, y) and the limits of the color scale are the same as the limits of the y-axis in C. (E) The 2DFES plot with RCs of QLID and Q. (F) The 2DFES plot with RCs of QNMP and Q. (G) The 2DFES plot with RCs of QCORE-N and QCORE-C shows that CORE-N folds before CORE-C in the predominant folding route.
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Related In: Results  -  Collection

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Show All Figures
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pcbi-1003938-g003: Contact clusters and free energy profiles of WT AKE.(A) The three contact clusters whose contacts form and break together during the folding and unfolding of WT AKE are projected onto their residues and shown on the structure: CORE-N (black; residues 1–29 and 68–117), LID (orange) and CORE-C (grey; residues 161–214). No contact cluster was found in NMP (green). (B) The contacts of the residues that form the clusters are shown in the same colors as in A. The intra-LID and intra-NMP contacts are enclosed in orange and green boxes and their contact location close to the diagonal underlines that LID and NMP are inserted domains. The CORE-N contacts are demarcated by the black box. (C–G) Reaction coordinates (RCs) for the folding free energy plots are defined as the fraction of native contacts formed in the whole protein (Q; all contacts, Fig. 2C), CORE-N (QCORE-N; black contacts in B), CORE-C (QCORE-C; grey contacts in B), CORE (QCORE; grey contacts, Fig. 2C), LID (QLID; orange contacts, Fig. 2C) and NMP (QNMP; green, Fig. 2C). The more folded a given region, the higher the value of the corresponding Q. (C) The scaled folding free energy (ΔG/kBTf) of WT AKE plotted as a function of Q. 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. (D) 2DFES plotted with RCs of QCORE and Q. The free energies are scaled by kBTf for all 2DFESs. The color indicates the height of the free energy at a given value of (x, y) and the limits of the color scale are the same as the limits of the y-axis in C. (E) The 2DFES plot with RCs of QLID and Q. (F) The 2DFES plot with RCs of QNMP and Q. (G) The 2DFES plot with RCs of QCORE-N and QCORE-C shows that CORE-N folds before CORE-C in the predominant folding route.
Mentions: Both the conformational transitions and the folding of AKE have previously been studied using different flavors of SBMs [18], [19], [32]. Here, we use a well-tested SBM [34] which uses only the C-α atom to represent the entire residue. This C-α SBM, which uses the folded structure (Fig. 1A, 2A, 2B) and its contact map (Fig. 1C, 2C) as inputs, is sufficient to capture the main changes in folding upon topological perturbation [35], [36] and is not intended for the detailed analysis of structural populations [2], [32]. We first validate the C-α SBM by performing folding simulations of WT AKE (Fig. 3) and show that these broadly agree with results from diverse ensemble experiments (HX-NMR [31], tryptophan fluorescence [24], and time-resolved FRET [26]–[30]).

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