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Investigating Domain Swapping by 19F-NMR, X-ray crystallography, and computations.

Lin Liu, Angela Gronenborn, Ivet Bahar, Leonardus Koharudin, and In-Ja Byeon


Energy diagram for domain-swapping of CV-N^(P51G) investigated by 19F-NMR

Among thousands of homo-oligomeric protein structures, there is a small but growing subset of 'domain-swapped' proteins. The term 'domain swapping,' originally coined by D. Eisenberg, describes a scenario in which two or more polypeptide chains exchange identical units for oligomerization. This type of assembly could play a role in disease-related aggregation and amyloid formation or as a specific mechanism for regulating function, and hence it is important to understand how proteins perform domain swapping. Although a lot of effort has been directed towards analyzing domain swapping, no unifying molecular mechanism of domain swapping has emerged to date. We compiled all domain-swapped protein structures in the PDB, performed a detailed examination of the common/different features of the chains in our collection, and summarized ideas about putative mechanisms.

Results from this analysis, for instance with respect to chain lengths, structural classification or amino acid composition, did not reveal any special properties associated with domain-swapped proteins or the exchanged domains. The diversity of sequences and architectures suggests that almost any protein may be capable of undergoing domain swapping and that domain swapping may be solely a specialized form of oligomer assembly. On the other side, structure-based computational analysis, i.e., GNM, on the monomeric conformations of our collection suggested that native contact topology information alone is not sufficient for uncovering hinge residues in our diverse set of domain-swapped proteins.

As the specific case for experimental investigation, we selected the protein cyanovirin-N (CV-N), a 101 amino acid cyanobacterial lectin that exhibits potent anti-HIV activity. CV-N contains only one tryptophan in its sequence and this tryptophan sits next to the hinge region for domain swapping; we therefore introduced 5-19F-tryptophan into the CV-N system, and used real-time 19F-NMR to monitor the conversion process between monomer and domain-swapped dimer. This novel method allowed us to determine the thermodynamic and kinetic determinants of CV-N domain swapping. The conversion between the monomeric and domain-swapped dimeric states was slow at room temperature, suggestion that conversion between the two states involves a transition over a high energy barrier. We determined this barrier height and found that it is of similar magnitude to the energy barrier for complete unfolding. Therefore, at least for CV-N, complete unfolding appears to be required for domain swapping.

We also carried out studies on additional domain-swapped oligomers. A domain-swapped trimer and a tetramer of CV-N have been prepared and we solved their structures by X-ray crystallography. This set of structures, along with the crystal structure of the domain-swapped dimer, provides insights into the potential mechanics of CV-N domain swapping. The results we obtained from these crystallographic investigations agree well with those from the 19F-NMR study.


Schematic representations of domain-swapped CV-N^(P51G) trimer (left) and tetramer (right)


Related publications: Liu, L, Byeon, IJ, Bahar, I, & Gronenborn, AM. (to be submitted). Investigating domain swapping by 19FNMR.

Liu, L & Gronenborn, AM. (in press). Domain swapping in proteins. Chapter in Comprehensive Biophysics, Ed. Daggett V., Elsevier.