PDBsum entry 2e4h

Structural protein

PDB id: 2e4h

Contents

Protein chain

74 a.a. *

Ligands

GLY-GLU-GLU-GLU-GLY-GLU-GLU-TYR

* Residue conservation analysis

PDB id:

2e4h

Name:

Structural protein

Title:

Solution structure of cytoskeletal protein in complex with tubulin tail

Structure:

Restin. Chain: a. Fragment: cap-gly domain 2, residues 203-300. Synonym: cytoplasmic linker protein 170 alpha-2, clip-170, reed- sternberg intermediate filament-associated protein, cytoplasmic linker protein 1. Engineered: yes. Tubulin alpha-ubiquitous chain. Chain: b.

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: clip-170. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693. Gene: a3-tubulin. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

M.Mishima,T.Hakoshima

Key ref:

M.Mishima et al. (2007). Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition. Proc Natl Acad Sci U S A, 104, 10346-10351. PubMed id: 17563362 DOI: 10.1073/pnas.0703876104

Date:

08-Dec-06 Release date: 28-Aug-07

Protein chain

P30622  (CLIP1_HUMAN) -  CAP-Gly domain-containing linker protein 1 from Homo sapiens

Seq: 1438 a.a.

Struc: 74 a.a.

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1073/pnas.0703876104

Proc Natl Acad Sci U S A 104:10346-10351 (2007)

PubMed id: 17563362

Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition.

M.Mishima, R.Maesaki, M.Kasa, T.Watanabe, M.Fukata, K.Kaibuchi, T.Hakoshima.

ABSTRACT

Cytoplasmic linker protein 170 (CLIP-170) is a prototype of the plus end-tracking proteins that regulate microtubule dynamics, but it is obscure how CLIP-170 recognizes the microtubule plus end and contributes to polymerization rescue. Crystallographic, NMR, and mutation studies of two tandem cytoskeleton-associated protein glycine-rich (CAP-Gly) domains of CLIP-170, CAP-Gly-1 and CAP-Gly-2, revealed positively charged basic grooves of both CAP-Gly domains for tubulin binding, whereas the CAP-Gly-2 domain possesses a more basic groove and directly binds the EExEEY/F motif of the C-terminal acidic-tail ends of alpha-tubulin. Notably, the p150(Glued) CAP-Gly domain that is furnished with a less positively charged surface only weakly interacts with the alpha-tubulin acidic tail. Mutation studies showed that this acidic sextette motif is the minimum region for CAP-Gly binding. The C-terminal zinc knuckle domains of CLIP-170 bind the basic groove to inhibit the binding to the acidic tails. These results provide a structural basis for the proposed CLIP-170 copolymerization with tubulin on the microtubule plus end. CLIP-170 strongly binds the acidic tails of EB1 as well as those of alpha-tubulins, indicating that EB1 localized at the plus end contributes to CLIP-170 recruitment to the plus end. We suggest that CLIP-170 stimulates microtubule polymerization and/or nucleation by neutralizing the negative charges of tubulins with the highly positive charges of the CLIP-170 CAP-Gly domains. Once CLIP-170 binds microtubule, the released zinc knuckle domain may serve to recruit dynein to the plus end by interacting with p150(Glued) and LIS1. Thus, our structures provide the structural basis for the specific dynein loading on the microtubule plus end.

Selected figure(s)

Figure 1.
Fig. 1. Crystal structures of the CAP-Gly-1 and CAP-Gly-2 domains. (a) Domain organization of the CLIP-170 dimer. The N-terminal CAP-Gly domains directly bind MT/tubulin. This binding is autoinhibited by the C-terminal zinc knuckle domains, which also serve to recruit dynein by interacting with p150^Glued and LIS1. (b) Ribbon drawings of the crystal structure of the CLIP-170 CAP-Gly-1 domain (Left), electrostatic potentials on the front molecular surface in the same molecular orientation (Center), and a 180° rotated image (Right). The region corresponding to the GKNDG motif is circled with yellow broken lines. (c) Same figures as in b but for the CLIP-170 CAP-Gly-2 domain. Electrostatic potentials of the front surfaces of the CAP-Gly domains of p150^Glued (PDB ID, 2HL3) (d), CYLD (1IXD) (e), and F53.43 (1LPL) (f).

Figure 3.
Fig. 3. Solution structure of the CAP-Gly-2–peptide complex. (a) A best-fit superimposition of the final 20 simulated annealing structures of the CLIP-170 CAP-Gly-2 domain (residues 212–281) bound to the 3-tubulin peptide (447–451) with the lowest energies is displayed. The CAP-Gly-2 domain backbone (cyan) and selected side chains (blue) are depicted with the peptide (magenta). (b) Close-up view of the hydrogen bonds (dotted lines) formed in the complex along the arrow in a (Right). The side chain of Phe-236 is omitted for clarity. (c) Pull-down assay of mutated CAP-Gly-2 domains with the 3-tubulin peptide suggests a critical role for Asn-253 in peptide binding.

Figures were selected by an automated process.