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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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Comparison of simulations with experimental refolding kinetics data.(A–B) WT AKE colored according to the contact clusters from Fig. 3A. NMP, which is not part of the clusters is shown in green as in Fig. 1A. Red (fast folding in experiment) and cyan (slow folding in experiment) spheres mark the positions of the C-α atoms of single tryptophan mutations used to study refolding kinetics using intrinsic fluorescence [24]. Brown spheres mark the positions of C-α atoms of FRET pairs used to study refolding kinetics by time resolved FRET [26]–[30]. Residues 73 and 86 were used in both experiments. Red lines connect the experimental early forming FRET distances while cyan lines connect the experimental late forming FRET distances. The probe residues lie in either CORE-N (black), CORE-C (grey) or NMP (green). (C) The native contacts of AKE colored similar to 3B, 4A and 4B: CORE-N (black), CORE-C (grey), LID (orange), NMP (green), and CORE-LID interface (yellow). The regions which fold early (CORE-N, black) and late (CORE-C, grey) during simulations (Fig. 3G) are demarcated by black and grey lines respectively. The red (early forming) and cyan (late forming) squares along the diagonal correspond to the single tryptophan mutants from A, B. Off diagonal red (early forming) and cyan (late forming) squares mark the FRET pairs joined by red and cyan lines in A, B.
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pcbi-1003938-g004: Comparison of simulations with experimental refolding kinetics data.(A–B) WT AKE colored according to the contact clusters from Fig. 3A. NMP, which is not part of the clusters is shown in green as in Fig. 1A. Red (fast folding in experiment) and cyan (slow folding in experiment) spheres mark the positions of the C-α atoms of single tryptophan mutations used to study refolding kinetics using intrinsic fluorescence [24]. Brown spheres mark the positions of C-α atoms of FRET pairs used to study refolding kinetics by time resolved FRET [26]–[30]. Residues 73 and 86 were used in both experiments. Red lines connect the experimental early forming FRET distances while cyan lines connect the experimental late forming FRET distances. The probe residues lie in either CORE-N (black), CORE-C (grey) or NMP (green). (C) The native contacts of AKE colored similar to 3B, 4A and 4B: CORE-N (black), CORE-C (grey), LID (orange), NMP (green), and CORE-LID interface (yellow). The regions which fold early (CORE-N, black) and late (CORE-C, grey) during simulations (Fig. 3G) are demarcated by black and grey lines respectively. The red (early forming) and cyan (late forming) squares along the diagonal correspond to the single tryptophan mutants from A, B. Off diagonal red (early forming) and cyan (late forming) squares mark the FRET pairs joined by red and cyan lines in A, B.

Mentions: Intrinsic fluorescence from single tryptophan (Trp) mutants has been used to study the refolding of AKE (24). These experiments show that regions of AKE near residues 41 (part of NMP), 86 and 73 (both part of CORE-N) (red spheres in Fig. 4A, B) fold faster than the region near residue 193 (part of CORE-C; cyan sphere in Fig. 4B). Thus, except for residue 41, the main folding route from our simulations where CORE-N folds before CORE-C (Fig. 3G) rationalizes the behaviour of the single Trp mutant fluorescence experiments. Further, we observe another route (∼10% of transitions) where CORE-C folds before CORE-N, in qualitative agreement with single molecule FRET data [2].


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)

Comparison of simulations with experimental refolding kinetics data.(A–B) WT AKE colored according to the contact clusters from Fig. 3A. NMP, which is not part of the clusters is shown in green as in Fig. 1A. Red (fast folding in experiment) and cyan (slow folding in experiment) spheres mark the positions of the C-α atoms of single tryptophan mutations used to study refolding kinetics using intrinsic fluorescence [24]. Brown spheres mark the positions of C-α atoms of FRET pairs used to study refolding kinetics by time resolved FRET [26]–[30]. Residues 73 and 86 were used in both experiments. Red lines connect the experimental early forming FRET distances while cyan lines connect the experimental late forming FRET distances. The probe residues lie in either CORE-N (black), CORE-C (grey) or NMP (green). (C) The native contacts of AKE colored similar to 3B, 4A and 4B: CORE-N (black), CORE-C (grey), LID (orange), NMP (green), and CORE-LID interface (yellow). The regions which fold early (CORE-N, black) and late (CORE-C, grey) during simulations (Fig. 3G) are demarcated by black and grey lines respectively. The red (early forming) and cyan (late forming) squares along the diagonal correspond to the single tryptophan mutants from A, B. Off diagonal red (early forming) and cyan (late forming) squares mark the FRET pairs joined by red and cyan lines in A, B.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003938-g004: Comparison of simulations with experimental refolding kinetics data.(A–B) WT AKE colored according to the contact clusters from Fig. 3A. NMP, which is not part of the clusters is shown in green as in Fig. 1A. Red (fast folding in experiment) and cyan (slow folding in experiment) spheres mark the positions of the C-α atoms of single tryptophan mutations used to study refolding kinetics using intrinsic fluorescence [24]. Brown spheres mark the positions of C-α atoms of FRET pairs used to study refolding kinetics by time resolved FRET [26]–[30]. Residues 73 and 86 were used in both experiments. Red lines connect the experimental early forming FRET distances while cyan lines connect the experimental late forming FRET distances. The probe residues lie in either CORE-N (black), CORE-C (grey) or NMP (green). (C) The native contacts of AKE colored similar to 3B, 4A and 4B: CORE-N (black), CORE-C (grey), LID (orange), NMP (green), and CORE-LID interface (yellow). The regions which fold early (CORE-N, black) and late (CORE-C, grey) during simulations (Fig. 3G) are demarcated by black and grey lines respectively. The red (early forming) and cyan (late forming) squares along the diagonal correspond to the single tryptophan mutants from A, B. Off diagonal red (early forming) and cyan (late forming) squares mark the FRET pairs joined by red and cyan lines in A, B.
Mentions: Intrinsic fluorescence from single tryptophan (Trp) mutants has been used to study the refolding of AKE (24). These experiments show that regions of AKE near residues 41 (part of NMP), 86 and 73 (both part of CORE-N) (red spheres in Fig. 4A, B) fold faster than the region near residue 193 (part of CORE-C; cyan sphere in Fig. 4B). Thus, except for residue 41, the main folding route from our simulations where CORE-N folds before CORE-C (Fig. 3G) rationalizes the behaviour of the single Trp mutant fluorescence experiments. Further, we observe another route (∼10% of transitions) where CORE-C folds before CORE-N, in qualitative agreement with single molecule FRET data [2].

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