FC0093
AF9, ANC1 Homology Domain (AHD)  -  AF4

Biological function
AF9 is one of the most common Mixed Lineage Leukemia (MLL) chromosomal translocation partner. AF9 is able to recruit and activate P-TEFb through recruitment of AF4 family members via the ANC1 homology domain (AHD). Disruption of the AF4-AF9 interaction results in necrotic cell death in several cell lines harboring MLL translocations.

Domain organization/sequence features
The ANC1 homology domain (AHD) interacts with both AF4 and Dot1L, which affects transcriptional elongation via histone methylation.

Structural evidence
The complex retains a significant amount of conformational entropy. ¹⁵N backbone relaxation data indicate conformational exchange in the complex on both ps-ns and ms-μs timescales. Several AF9 loops, particularly those near the AF4 peptide, are involved in conformational exchange. The loop from 515-519 shows fast timescale dynamics, and 534-542 show ms-μs exchange. The NOESY spectra suggest significant internal dynamics in the core of the protein which are reflected in the backbone. The HSQC spectrum of AHD is very similar in the presence of other binding partners, such as hPC3, Dot1L and BCoR, suggesting that their binding takes place in a mutually exclusive manner.

Biochemical evidence
By fluorescence anisotropy the affinity for AF4 is extremely high (K_D = 0.17 ± 0.05 nM). The significant amount of conformational entropy may partially compensate for the loss of conformational entropy during coupled folding and binding. Similarly high affinity was observed for Dot1L (1.6 ± 0.3 nM) and BCoR (32 ± 20 nM), however the peptide from hPC3 binds with much lower affinity (K_D > 0.9 μM). The hPC3 sequence lacks a buried valine residue which is part of the consensus sequence, and introduction V335 in the hPC3 peptide decreased the dissociation constant between AF9 and hPC3 to 8.7 ± 0.7 nM.

Structure/Mechanism
Mutual, synergistic folding: the aliphatic side chains of AF4 form an integral part of the hydrophobic core of the AF4-AF9 complex, generating an extensive hydrophobic interface.

Mechanism category
flexibility modulation

Significance
Fuzziness of the complex allows exchange between binding partners in response to changes in local concentrations or post-translational modifications, which may be essential to dynamic transcriptional control.

Medical relevance
Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene are responsible for a subset of acute leukemias characterized by poor prognosis and early relapse. The importance of the AF9 AHD in both transcriptional regulation and in acute leukemia has been established.

Submitted by
John Bushweller   jhb4v@virginia.edu, bileach@scripps.edu