PDBsum entry 2jy8

Protein binding

PDB id

2jy8

Contents

			Protein chain

					52 a.a.

References listed in PDB file

Key reference

Title

Ubiquitin recognition by the ubiquitin-Associated domain of p62 involves a novel conformational switch.

Authors

J.Long, T.R.Gallagher, J.R.Cavey, P.W.Sheppard, S.H.Ralston, R.Layfield, M.S.Searle.

Ref.

J Biol Chem, 2008, 283, 5427-5440. [DOI no: 10.1074/jbc.M704973200]

PubMed id

18083707

Abstract

The p62 protein functions as a scaffold in signaling pathways that lead to activation of NF-kappaB and is an important regulator of osteoclastogenesis. Mutations affecting the receptor activator of NF-kappaB signaling axis can result in human skeletal disorders, including those identified in the C-terminal ubiquitin-associated (UBA) domain of p62 in patients with Paget disease of bone. These observations suggest that the disease may involve a common mechanism related to alterations in the ubiquitin-binding properties of p62. The structural basis for ubiquitin recognition by the UBA domain of p62 has been investigated using NMR and reveals a novel binding mechanism involving a slow exchange structural reorganization of the UBA domain to a "bound" non-canonical UBA conformation that is not significantly populated in the absence of ubiquitin. The repacking of the three-helix bundle generates a binding surface localized around the conserved Xaa-Gly-Phe-Xaa loop that appears to optimize both hydrophobic and electrostatic surface complementarity with ubiquitin. NMR titration analysis shows that the p62-UBA binds to Lys 48-linked di-ubiquitin with approximately 4-fold lower affinity than to mono-ubiquitin, suggesting preferential binding of the p62-UBA to single ubiquitin units, consistent with the apparent in vivo preference of the p62 protein for Lys 63-linked polyubiquitin chains (which adopt a more open and extended structure). The conformational switch observed on binding may represent a novel mechanism that underlies specificity in regulating signalinduced protein recognition events.



	Figure 1. FIGURE 1. Sequence and structure of the p62 protein and its UBA domain. Schematic representation of the p62 protein and its domain structure is shown (A). The sequence of the C-terminal UBA domain is shown expanded with the numbering from the full-length protein shown (residues 387–436). The position of the conserved Met-Gly-Phe-Ser loop is underlined; Gly^410 and Gly^411 correspond to the double glycine insertion (B). The secondary structure content of the UBA domain identified in the unbound form by NMR is shown in C. NMR structure of the p62-UBA domain shows the position of mutated residues associated with Paget disease of bone (D), and a ribbon structure of the UBA domain shows the packing of core hydrophobic residues Met^401, Leu^417, Ala^427, and Ile^431 (E).		Figure 3. FIGURE 3. Structural analysis of the p62 UBA domain and the binding surfaces of the UBA and mUb. Chemical shift perturbations are mapped to the surface of Ub on a linear scale of white to red (largest) (A), as described by the data in Fig. 2B. B, surface representation of the structure of the p62-UBA domain in the Ub-bound state showing the surface charge distribution (acidic, red, and basic, blue) and hydrophobicity (white). The structure is viewed toward the Met-Gly-Phe-Ser loop region. C, identical orientation and representation of the UBA domain of p62 showing secondary fast exchange chemical shift perturbations (red) indicative of binding interactions with mUb. These binding perturbations correlate well with the hydrophobic surface shown in B, comprising residues within loop 1 and the C terminus of helix 3. Ribbon diagram shows NMR structures of the unbound UBA domain (D) and the bound form in the presence of 6 eq of mUb (E). The structural statistics are shown in Table 1. In the unbound state the environment of the side chain of Gln^400 is defined by NOEs with residues Phe^406, Ile^424, and Leu^428 (F); in contrast, the repacking of the helices in the bound state shows that Gln^400 is close in space to Trp^412, Leu^416, and Leu^417 (G). In this conformation the side chains of Phe^406, Ile^424, and Leu^428 are more remote from Gln^400. A schematic representation of the two structures is shown to illustrate more clearly the structural reorganization and repacking of the three helices that occurs (H). The cylindrical arrows represent the orientation and polarity of the helices. H, free (blue) and bound (red) structures are shown with a common alignment of helices 1 and 2. These structures are superimposed in I, showing the different positions and orientations of helix 3 in the two forms.

PROCHECK

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