Abstract
Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.
Highlights
Processes such as point mutation, gene duplication and fusion, recombination and circular permutation drive evolution
The cpSAP97 PDZ2 protein structure has the typical protein95/Discs large/Zonula Occludens-1 (PDZ) domain fold with six b-strands (b1 to b6) and two ahelices (a1 and a2) (Figure 1B)
It has been argued that changes in folding pathway due to circular permutation depend on the folding nucleus; a diffuse folding nucleus covering most of the protein is less likely to change the folding pathway compared to a regional compact nucleus [9,40]
Summary
Processes such as point mutation, gene duplication and fusion, recombination and circular permutation drive evolution. Among the handful of studies on isolated protein domains that have been published, circular permutation often results in more complex kinetic folding mechanisms than for the wild type [2,3,4,5] and sometimes population of low energy intermediates [6,7,8]. Otzen and Fersht suggested that folding of protein domains with diffuse folding nuclei are more likely to be unaffected by circular permutation. To learn more about how circular permutation affects folding pathways, we analyzed a protein domain with a relatively complex folding pathway, namely the second Postsynaptic density protein95/Discs large/Zonula Occludens-1 (PDZ) domain from synapse associated protein 97 (SAP97). PDZ domains are usually part of such multi domain proteins and have important roles in molecular recognition
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