PDBsum entry 3hqh

Ligase

PDB id: 3hqh

Contents

Protein chain

134 a.a. *

Ligands

ALA-SER-ALA-ASP-SER-THR-THR-GLU-GLY

Metals

_ZN ×3

Waters ×48

* Residue conservation analysis

PDB id:

3hqh

Name:

Ligase

Title:

Structures of spop-substrate complexes: insights into molecular architectures of btb-cul3 ubiquitin ligases: spopmathx- macroh2asbcpep1

Structure:

Speckle-type poz protein. Chain: a. Engineered: yes. Macroh2a. Chain: m. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: spop. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Other_details: synthetic peptide

Resolution:

2.30Å R-factor: 0.238 R-free: 0.276

Authors:

M.Zhuang,B.A.Schulman

Key ref:

M.Zhuang et al. (2009). Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases. Mol Cell, 36, 39-50. PubMed id: 19818708 DOI: 10.1016/j.molcel.2009.09.022

Date:

06-Jun-09 Release date: 20-Oct-09

Protein chain

O43791  (SPOP_HUMAN) -  Speckle-type POZ protein from Homo sapiens

Seq: 374 a.a.

Struc: 134 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1016/j.molcel.2009.09.022

Mol Cell 36:39-50 (2009)

PubMed id: 19818708

Structures of SPOP-substrate complexes: insights into molecular architectures of BTB-Cul3 ubiquitin ligases.

M.Zhuang, M.F.Calabrese, J.Liu, M.B.Waddell, A.Nourse, M.Hammel, D.J.Miller, H.Walden, D.M.Duda, S.N.Seyedin, T.Hoggard, J.W.Harper, K.P.White, B.A.Schulman.

ABSTRACT

In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here, we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC) and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call "3-box" due to its facilitating Cul3 binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3 and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.

Selected figure(s)

Figure 4.
Figure 4. SPOP^BTB+ Forms a 2:2 Dimer with Cul3 N-Terminal Domain
(A) Left, overall view of the SPOP^BTB+ dimer, with protomers in cyan (A) and red (B). Right, close-up view of dimer interface rotated 90° in x.
(B) Equilibrium AUC of SPOP^BTB+ + Cul3^NTD. Samples at 1.0 to 8.8 μM centrifuged at 8,000 (red), 12,000 (blue), and 16,000 (black) rpm (4°C). Lines show global nonlinear least-squares best-fit of all data sets/concentrations/speeds to a heterogeneous association model describing a 2:2 SPOP^BTB+:Cul3^NTD complex (MW 127.1 kDa) with indicated K[D] value. For clarity, only the 3 μM sample is shown.
(C) AUC of L186D, L190D, L193D, and I217K mutant SPOP^BTB+ + Cul3^NTD performed as in (B). Lines show global nonlinear least-squares best-fit of all data sets/concentrations/ speeds to a heterogeneous association model describing a 1:1 SPOP^BTB+ (mutant):Cul3^NTD complex (MW 63.6 kDa) with the indicated K[D] value. For clarity, only the 2.0 μM sample is shown.
(D) Western blots of SPOP^MATH-BTB+-mediated ubiquitination detecting His-Puc for wild-type and L186D, L190D, L193D, and I217K (dimer-defective) mutant SPOP.

Figure 7.
Figure 7. A 1:2 Substrate Complex with the SPOP-Cul3 Ubiquitin Ligase
(A) Velocity AUC of SPOP^MATH-BTB/3-box + Puc^1–390 at 20°C and 60,000 rpm fit to a continuous distribution model c(s). Two peaks indicate molecular weights of 110 kDa and 39 kDa, corresponding to the 1:2 Puc:SPOP^MATH-BTB/3-box complex (MW[calc] of 112.5 kDa) and excess free Puc (MW[calc] of 42.1 kDa).
(B) Equilibrium AUC of a sample as in (A). Samples at 1–6 μM centrifuged at 8,000 (red), 12,000 (blue), and 16,000 (black) rpm (4°C). Lines show global nonlinear least-squares best-fit of all data sets/concentrations/speeds to a heterogeneous association model with two species, 2:1 SPOP^MATH-BTB/3-box:Puc + Puc. For clarity, only the 1.1 μM sample is shown.
(C) Overall structure of SPOP^MATHx-MacroH2A^SBC (pep2). Two isolated MATH domains (chain A, cyan; chain B, pink) bind a single-substrate peptide (green) at two suboptimal SBC sites.
(D) Schematic view of a SPOP-Cul3 ubiquitin ligase bound to a single substrate. Substrate is shown in gray, with SBCs in green and ubiquitin-acceptor lysines as Ks. The two protomers of the dimeric SPOP complex are shown in cyan and red, with each BTB/3-box bound near the N terminus of an elongated Cul3 (olive) activated with NEDD8 (orange) near the C terminus. E2-bound Rbx1 RING domains are shown flexibly tethered to the Cul3 C-terminal domains. The high degree of conformational flexibility may allow substrates with a range of SBC configurations to be polyubiquitinated at multiple sites.

The above figures are reprinted by permission from Cell Press: Mol Cell (2009, 36, 39-50) copyright 2009.

Figures were selected by an automated process.