Abstract
PDZ domains are the most prominent biological structural domains involved in protein-protein interactions in the human cell. The second PDZ domain of the protein tyrosine phosphatase BL (PDZ2) interacts and binds the C-termini of the tumour suppressor protein APC and of the LIM domain-containing protein RIL. One isoform of PDZ2 (PDZ2as) involves an alternative spliced form that exhibits an insertion of 5 residues in a loop. PDZ2as abrogates binding to its partners, even if the insertion is directly located in its binding pocket. Here, we investigate the folding and function of PDZ2as, in comparison to the previously characterized PDZ2 domain. Data reveal that, whilst the thermodynamic stability of PDZ2as appears as nearly identical to that of PDZ2, the insertion of 5 amino acids induces formation of some weak transient non-native interactions in the folding transition state, as mirrored by a concomitant increase of both the folding and unfolding rate constants. From a functional perspective, we show that the decrease in affinity is caused by a pronounced decrease of the association rate constants (by nearly ten fold), with no effect on the microscopic dissociation rate constants. The results are briefly discussed in the context of previous work on PDZ domains.
Highlights
Understanding the effect of alternative splicing in the folding and function of the second PDZ from Protein Tyrosine Phosphatase-BL
Despite it has been previously observed that the b2–b3 loop may have a role in fine tuning the affinity of PDZ domains to their target[29,30], the remarkable change in affinity induced by the alternative splicing is somewhat surprising, the binding pocket being physically located at the interface between helix a2 and www.nature.com/scientificreports strand b2
On the contrary of what previously suggested by NMR experiments[28], it appears that the apparent stability of pWT43 PDZ2as, defined as the change in free energy between the native and the denatured state, is nearly identical to that observed for pWT43 PDZ2, corresponding to a value of 3.6 6 0.2 kcal mol[21 34]
Summary
Understanding the effect of alternative splicing in the folding and function of the second PDZ from Protein Tyrosine Phosphatase-BL. Despite it has been previously observed that the b2–b3 loop may have a role in fine tuning the affinity of PDZ domains to their target[29,30], the remarkable change in affinity induced by the alternative splicing is somewhat surprising, the binding pocket being physically located at the interface between helix a2 and www.nature.com/scientificreports strand b2. This finding appears to confirm the existence of long range effects in PDZ domains. The conformational changes induced by the insertion of the 5 residues in PDZ2as appear very similar to those observed between the free state of PDZ2 and its bound state to APC (Figure 1)[10,28]
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