Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.

Structural and thermodynamic characterization of pre- and postpolymerization states in the F4 fimbrial subunit FaeG.

Enterotoxigenic Escherichia coli expressing F4 fimbriae are the major cause of porcine colibacillosis and are responsible for significant death and morbidity in neonatal and postweaned piglets. Via the chaperone-usher pathway, F4 fimbriae are assembled into thin, flexible polymers mainly composed of the single-domain adhesin FaeG. The F4 fimbrial system has been labeled eccentric because the F4 pilins show some features distinct from the features of pilins of other chaperone-usher-assembled structures. In particular, FaeG is much larger than other pilins (27 versus approximately 17 kDa), grafting an additional carbohydrate binding domain on the common immunoglobulin-like core. Structural data of FaeG during different stages of the F4 fimbrial biogenesis process, combined with differential scanning calorimetry measurements, confirm the general principles of the donor strand complementation/exchange mechanisms taking place during pilus biogenesis via the chaperone-usher pathway.

15N NMR relaxation data reveal significant chemical exchange broadening in the alpha-domain of human α-lactalbumin

Human alpha-lactalbumin (alpha-LA), a 123-residue calcium-binding protein, has been studied using (15)N NMR relaxation methods in order to characterize backbone dynamics of the native state at the level of individual residues. Relaxation data were collected at three magnetic field strengths and analyzed using the model-free formalism of Lipari and Szabo. The order parameters derived from this analysis are generally high, indicating a rigid backbone. A total of 46 residues required an exchange contribution to T(2); 43 of these residues are located in the alpha-domain of the protein. The largest exchange contributions are observed in the A-, B-, D-, and C-terminal 3(10)-helices of the alpha-domain; these residues have been shown previously to form a highly stable core in the alpha-LA molten globule. The observed exchange broadening, along with previous hydrogen/deuterium amide exchange data, suggests that this part of the alpha-domain may undergo a local structural transition between the well-ordered native structure and a less-ordered molten-globule-like structure.

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATPBinding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse Pgp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.