Structural analysis of histone deacetylase 8 mutants associated with Cornelia de Lange Syndrome spectrum disorders

Abstract

Cornelia de Lange Syndrome (CdLS) and associated spectrum disorders are characterized by one or more congenital anomalies including distinctive facial features, upper limb abnormalities, intellectual disability, and other symptoms. The molecular genetic basis of CdLS is linked to defects in cohesin, a protein complex that functions in sister chromatid cohesion, chromatin organization, and transcriptional regulation. Histone deacetylase 8 (HDAC8) plays an important role in cohesin function by catalyzing the deacetylation of SMC3, which is required for efficient recycling of the cohesin complex. Missense mutations in HDAC8 have been identified in children diagnosed with CdLS spectrum disorders, and here we outline structure-function relationships for four of these mutations. Specifically, we report the 1.50 Å-resolution structure of the I45T HDAC8-suberoylanilide hydroxamic acid complex, the 1.84 Å-resolution structure of E66D/Y306F HDAC8 complexed with a peptide assay substrate, and the 2.40 Å-resolution structure of G320R HDAC8 complexed with the inhibitor M344. Additionally, we present a computationally generated model of D176G HDAC8. These structures illuminate new structure-function relationships for HDAC8 and highlight the importance of long-range interactions in the protein scaffold that can influence catalytic function.

Keywords: Birth defect; Cohesin; X-ray crystallography; Zinc enzyme.

Figure 1.

(A) Cohesin structural proteins SMC1A and SMC3 associate through their hinge domains and encircle sister chromatids during cell division; RAD21 associates with the ATPase domains of SMC1A and SMC3, and STAG1 or STAG2 binds to RAD21 to stabilize cohesin closure. ESCO1 and ESCO2 are responsible for acetylation of K105 and K106 in the ATPase domain of SMC3, and HDAC8 is responsible for SMC3 deacetylation. Reproduced from Deardorff et al., 2016. (B) The I45T, E66D, D176G, and G320R mutations in HDAC8 have been identified in children diagnosed with Cornelia de Lange Syndrome (CdLS) spectrum disorders. These residues are mapped onto the structure of Y306F HDAC8 complexed with the assay substrate Ac-RHKAcKAc-AMC (Ac = acetyl, Kac = acetyllysine, AMC = aminomethylcoumarin) (PDB 2V5W). Color code: HDAC8, yellow, CdLS mutations, red; Zn²⁺, small gray sphere; K⁺ ions, large orange spheres; substrate, stick figure with C = gray, N = blue, O = red.

Figure 2.

Polder omit map of SAHA bound in the active site of I45T HDAC8 (PDB 7JVU; monomer A, contoured at 4.0 σ). Atoms are color-coded as follows: C = yellow (monomer A), light gray (monomer B), gray (SAHA, monomer A), or dark gray (SAHA, monomer B), N = blue, O = red, S = light yellow, Zn²⁺ = gray sphere, and solvent = small red spheres. Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively.

Figure 3.

(A) Polder omit map of T45 in the I45T HDAC8–SAHA complex (PDB 7JVU; monomer A, contoured at 3.5 σ). Atoms are color-coded as follows: C = yellow, N = blue, O = red. Hydrogen bond interactions are indicated by dashed black lines, respectively. (B) Superposition of the I45T HDAC8–SAHA complex shown in (A) with monomer B in the same structure (color-coded with C = orange). (C) Superposition of the I45T HDAC8–SAHA complex shown in (A) with the wild-type HDAC8–SAHA complex (PDB 1T69). Atoms are color-coded as follows: C = yellow (I45T HDAC8) or gray (wild-type HDAC8), N = blue, O = red.

Figure 4.

(A) Polder omit map of the substrate in the E66D/Y306F HDAC8–Ac-RHKAcKAc-AMC complex (PDB 7JVV; monomer A, contoured at 5.0 σ). Atoms are color-coded as follows: C = yellow (monomer A), light gray (monomer B), or gray (substrate), N = blue, O = red, Zn²⁺ = gray sphere, and solvent = small red spheres. Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively. (B) Superposition of the E66D/Y306F HDAC8–Ac-RHK_AcK_Ac-AMC complex shown in (A) with the structure of the Y306F HDAC8–Ac-RHK_AcK_Ac-AMC complex (PDB 2V5W).

Figure 5.

(A) Polder omit map of D66 in the E66D/Y306F HDAC8–RHKACKAC-AMC complex (PDB 7JVV; monomer A, contoured at 3.0 σ). Atoms are color-coded as follows: C = yellow (monomer A) or light gray (monomer B), N = blue, O = red, and solvent = small red spheres; GOL = glycerol. Hydrogen bond interactions are indicated by dashed black lines. (B) Superposition of the E66D/Y306F HDAC8–Ac-RHK_AcK_Ac-AMC complex shown in (A) with the structure of the Y306F HDAC8–Ac-RHK_AcK_Ac-AMC complex (PDB 2V5W). Atoms are color-coded as follows: C = yellow (E66D/Y306F HDAC8) or gray (wild-type HDAC8), N = blue, O = red.

Figure 6.

Polder omit map of the inhibitor M344 in the G320R HDAC8–M344 complex (PDB 7JVW; monomer A, contoured at 3.5 σ). Atoms are color-coded as follows: C = yellow (monomer A), light gray (monomer B), gray (inhibitor), or dark gray (inhibitor bound to monomer B), N = blue, O = red, Zn²⁺ = gray sphere, and solvent = small red spheres. Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively.

Figure 7.

(A) Polder omit map of R320 in the G320R HDAC8–M344 complex (PDB 7JVW; monomer A, contoured at 3.5 σ). Atoms are color-coded as follows: C = yellow, N = blue, O = red, and solvent = small red spheres. Hydrogen bond interactions are indicated by dashed black lines. (B) Superposition of the G320R HDAC8–M344 complex shown in (A) with the wild-type HDAC8–M344 complex (PDB 1T67). Atoms are color-coded as follows: C = yellow (G320R HDAC8) or gray (wild-type HDAC8), N = blue, O = red.

Figure 8.

Superposition of the computationally-derived model of D176G HDAC8 and the experimentally-determined structure of D176A/Y306F HDAC8 complexed with an assay substrate (PDB 5DC7). Atoms are color-coded as follows: C = yellow (D176G HDAC8) or gray (D176A HDAC8), N = blue, O = red, Zn²⁺ = gray sphere.