PDBsum entry 1zc4

Signaling protein

PDB id: 1zc4

Contents

Protein chains

173 a.a.

113 a.a.

Ligands

GNP ×2

Metals

_MG ×2

Waters ×153

References listed in PDB file

Key reference

Title

Exo84 and sec5 are competitive regulatory sec6/8 effectors to the rala gtpase.

Authors

R.Jin, J.R.Junutula, H.T.Matern, K.E.Ervin, R.H.Scheller, A.T.Brunger.

Ref.

EMBO J, 2005, 24, 2064-2074. [DOI no: 10.1038/sj.emboj.7600699]

PubMed id

15920473

Abstract

The Sec6/8 complex, also known as the exocyst complex, is an octameric protein complex that has been implicated in tethering of secretory vesicles to specific regions on the plasma membrane. Two subunits of the Sec6/8 complex, Exo84 and Sec5, have recently been shown to be effector targets for active Ral GTPases. However, the mechanism by which Ral proteins regulate the Sec6/8 activities remains unclear. Here, we present the crystal structure of the Ral-binding domain of Exo84 in complex with active RalA. The structure reveals that the Exo84 Ral-binding domain adopts a pleckstrin homology domain fold, and that RalA interacts with Exo84 via an extended interface that includes both switch regions. Key residues of Exo84 and RalA were found that determine the specificity of the complex interactions; these interactions were confirmed by mutagenesis binding studies. Structural and biochemical data show that Exo84 and Sec5 competitively bind to active RalA. Taken together, these results further strengthen the proposed role of RalA-regulated assembly of the Sec6/8 complex.

Figure 2.
Figure 2 Structure of the Exo84-RBD:RalA complex. (A) Ribbon diagram of the Exo84-RBD:RalA complex. Exo84-RBD is colored in red. RalA is colored in green, except that switch I (38 -50) and switch II (69 -85) are highlighted in orange. The secondary structures of RalA are numbered in a sequential order. The GMPPNP is shown in a ball-and-stick representation and the Mg2+ is shown as a gray sphere. A close-up view of the boxed region is shown in panel B, which is superimposed with a portion of electron density map. (B) Representative portion of a [A]-weighted 2F[o]-F[c] electron density map (contoured at 1.0 ) overlaid with the final refined model. The Exo84 and RalA molecules are colored as in panel A and the selected residues are shown in a ball-and-stick representation. (C) Ribbon representation of the Exo84-RBD structure. The secondary structure elements are numbered in a sequential order. (D) Exo84-RBD:RalA complex forms a two-fold symmetry related dimer in the crystal. The Exo84-RBD molecules are red and cyan, while the RalA molecules are green and light purple, respectively. Also shown are the two GMPPNP molecules. The putative phospholipid-binding sites are indicated by green oval circles.

Figure 4.
Figure 4 Exo84 and Sec5 have overlapping binding sites on the active RalA. (A) Superposition of the Exo84-RBD:RalA and the Sec5-RBD:RalA complexes. RalA is green in the Exo84-RBD:RalA complex and purple when in complex with Sec5-RBD. Exo84-RBD and Sec5-RBD are colored in red and blue, respectively. The two RalA molecules are superimposed using all equivalent C atoms except for residues in the two switch regions. Note that Exo84 and Sec5 cannot bind to RalA simultaneously. Close-up views of the areas that are indicated by red and blue boxes are shown in panels B and C, respectively. (B) Close-up view of the complex interface around RalA switch II where significantly different RalA conformations were observed between the two complexes. Shown are the five RalA residues in this region that directly contact Exo84-RBD. The molecules are colored as in panel A. (C) Close-up view of the Sec5-RBD:RalA interface. Shown are the five RalA residues that form hydrogen bonds with Sec5-RBD. The color scheme is the same as in panels A and B. (D) Molecular surface of RalA when it is in complex with Exo84-RBD. The RalA residues that exclusively contact Exo84-RBD are colored red, the residues that only bind Sec5-RBD are colored blue and the residues that are involved in interactions with both effectors are colored in orange.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (2005, 24, 2064-2074) copyright 2005.