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J. Mol. Biol. 330, 473-484 (2003)
An NMR solution structure of the C-terminal domain of human centrin (caltractin) isoform 2 (Cen2) has recently been reported by Crasecu and coworkers1 (PDB accession code 1M39). An unusual construct (LC-HsCen2) was used for these studies, which contains an additional N-terminal extension that includes the linker between the N- and C-terminal domains and a substantial portion of helix IV from the N-terminal domain. The presence of the "extra helix" greatly complicates the interpretation of their results for several reasons, of which the most significant is the "extra helix" in isolation has unsatisfied packing interactions and thus folds back on, and interacts with, the calcium-activated C-terminal domain. The authors claim this structure serves as a model for centrin-target interactions, but our analysis shows that there are significant problems with the structure of LC-HsCen2 that strongly imply the proposed model is inaccurate.
The key problem with the LC-HsCen2 structure is that it is not a high resolution structure due to the lack of sufficient experimental constraints (Table S1). For example, there are 248 long range distance constraints for LC-HsCen2 versus 429 for the CRC-C/K19 complex. The limited number of experimental data results in low precision overall, which is reflected in the high RMSDs (Table S1). The most significant limitation in the experimental data is that NMR signals from the fourth helix of the Cen2-C domain are exchange broadened, which precluded proper structural definition of this helix and the packing of this helix against the other helices. In comparing this structure to CRC-C and the C-terminal domain of CaM (CaM-C), we find that the two high resolution structures are very similar to each other, whereas LC-HsCen2 is significantly different. For example, the similarity of interhelical angles for CRC-C and CaM-C contrasts sharply with the differences when comparing these to LC-HsCen2 (Table S2). Given the sequence homologies, there is no reason for LC-HsCen2 to be different.
Detailed comparative analyses are required to understand protein-target interactions, and high resolution structures are absolutely essential to achieving this goal. Clearly, the structure of LC-HsCen2 does not meet this criterion. Nonetheless, we proceeded to carry out a comparative analysis of the LC-HsCen2 structure and found it is not likely to be a good model for target interactions. In this model, the "extra helix" at the N-terminus of the construct represents the target. This helix is found to bind in a completely different orientation than K19 in the CRC-C complex or R20 and other CaM-binding peptides in their corresponding CaM complexes (Table S3). LC-HsCen2 has a fundamental problem because the "extra helix" is tethered to the Cen2-C domain and is sterically restricted from accessing the full range of potential orientations with respect to the C-terminal domain. An additional factor is the sequence of the "extra helix" is not particularly complementary to the target binding surface of the HsCen2 C-terminal domain, whereas there is a finely tuned match between protein and target found in the CRC-C and CaM complexes.
In summary, the key finding from the study by Craescu and coworkers is that the HsCen2-C domain occupies an open conformation in the calcium-bound state and this conclusion is fully valid. But the authors' claim that this system serves as a model for centrin-target complexes is highly questionable. Detailed analysis of this structure and comparisons to the structures of CRC-C and CaM complexes strongly imply such a model is inaccurate.
1. E. Matei, S. Miron, Y. Blouquit, P. Duchambon, I. Durussel, J.A. Cox and C.T. Craescu, C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain. Biochemistry 42 (2003), pp. 1439-1450.