Abstract
AF4/FMR2 family member 4 (AFF4) is the scaffold protein of the multisubunit super-elongation complex, which plays key roles in the release of RNA polymerase II from promoter-proximal pausing and in the transactivation of HIV-1 transcription. AFF4 consists of an intrinsically disordered N-terminal region that interacts with other super-elongation complex subunits and a C-terminal homology domain (CHD) that is conserved among AF4/FMR2 family proteins, including AFF1, AFF2, AFF3, and AFF4. Here, we solved the X-ray crystal structure of the CHD in human AFF4 (AFF4-CHD) to 2.2 Å resolution and characterized its biochemical properties. The structure disclosed that AFF4-CHD folds into a novel domain that consists of eight helices and is distantly related to tetratrico peptide repeat motifs. Our analyses further revealed that AFF4-CHD mediates the formation of an AFF4 homodimer or an AFF1-AFF4 heterodimer. Results from fluorescence anisotropy experiments suggested that AFF4-CHD interacts with both RNA and DNA in vitro Furthermore, we identified a surface loop region in AFF4-CHD as a substrate for the P-TEFb kinase cyclin-dependent kinase 9, which triggers release of polymerase II from promoter-proximal pausing sites. In conclusion, the AFF-CHD structure and biochemical analyses reported here reveal the molecular basis for the homo- and heterodimerization of AFF proteins and implicate the AFF4-CHD in nucleic acid interactions. The high conservation of the CHD among several other proteins suggests that our results are also relevant for understanding other CHD-containing proteins and their dimerization behavior.
Highlights
AF4/FMR2 family member 4 (AFF4) is the scaffold protein of the multisubunit super-elongation complex, which plays key roles in the release of RNA polymerase II from promoter-proximal pausing and in the transactivation of HIV-1 transcription
The crystal structure reveals that AFF4-C-terminal homology domain (CHD) folds into eight ␣-helices, with ␣1–␣4, ␣7, and one-half of ␣6, forming a structure with a concave side, which is filled by the last helix ␣8 and covered by the N-terminal tail region that folds onto ␣8 (Fig. 1B)
The results showed that the AFF4-CHD partially aligns with the tetratrico peptide repeat (TPR) motif– containing proteins, such as anaphase-promoting complex subunit 5 (5G05-O) [30], 14-3-3 protein ␥ (6GKG-F) [31], G-protein-signaling modular 2 (4JHR-B) [32], and 14-3-3 protein /␣ (6HEP-B) [33] with Z scores of 9.1, 8.9, 8.9, and 8.8, respectively
Summary
AF4/FMR2 family member 4 (AFF4) is the scaffold protein of the multisubunit super-elongation complex, which plays key roles in the release of RNA polymerase II from promoter-proximal pausing and in the transactivation of HIV-1 transcription. AFF4 consists of an intrinsically disordered N-terminal region that interacts with other super-elongation complex subunits and a C-terminal homology domain (CHD) that is conserved among AF4/FMR2 family proteins, including AFF1, AFF2, AFF3, and AFF4. In addition to the N-terminal disordered region, AFF4 shares a highly conserved C-terminal homology domain (CHD) with other AF4/FMR family proteins, including AFF1, AFF2, and AFF3. The latter proteins are scaffolds of SEC-L2 and SEC-L3 complexes that contain P-TEFb and AF9/ENL but lack ELL/ EAF subunits. Our work contributes to the structural characterization of SEC, which is a key player in gene regulation in eukaryotic cells
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