The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2 beta

Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.publishedVersio

Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A

<div><p>Bromodomain and extra-terminal (BET) family of proteins are one of the major readers of epigenetic marks and an important target class in oncology and other disease areas. The importance of the BET family of proteins is manifested by the explosion in the number of inhibitors against these targets that have successfully entered clinical trials. One important BET family member is bromodomain containing protein 4 (BRD4). Structural and biophysical studies of BRD4 are complicated by its tertiary-structure consisting of two bromodomains connected by a flexible inter-domain linker of approximately 180 amino acids. A detailed understanding of the interplay of these bromodomains will be key to rational drug design in BRD4, yet there are no reported three-dimensional structures of the multi-domain BRD4 and NMR studies of the tandem domain are hampered by the size of the protein. Here, we present a method for rapid Sortase A-mediated segmental labelling of the individual bromodomains of BRD4 that provides a powerful strategy that will enable NMR studies of ligand-bromodomain interactions with atomic detail. In our labelling strategy, we have used U-[²H,¹⁵N]-isotope labelling on the C-terminal bromodomain with selective introduction of ¹³CH₃ methyl groups on Ile (δ1), Val and Leu, whereas the N-terminal bromodomain remained unlabelled. This labelling scheme resulted in significantly simplified NMR spectra and will allow for high-resolution interaction, structure and dynamics studies in the presence of ligands.</p></div

Schematic illustration of segmental labelling on BRD4(1, 2) using SrtA.

<p>Isotopically labelled BRD4^C (orange) was ligated to unlabelled BRD4^NL (blue) to give a segmentally labelled BRD4(1, 2) fusion. TEV protease cleavage and Sortase A ligation reactions were carried out separately. The two step reaction mechanisms of SrtA is also illustrated here. Step I involves an initial nucleophilic attack of the C-terminal LPDTG recognition sequence by Cys184 of SrtA. In step II there is a subsequent nucleophilic attack by the N-terminal Gly of the BRD4^C construct.</p

Bromodomain Interactions with Acetylated Histone 4 Peptides in the BRD4 Tandem Domain : Effects on Domain Dynamics and Internal Flexibility

The bromodomain and extra-terminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Like other BET proteins, BRD4 contains two bromodomains, BD1 and BD2, that can interact cooperatively with target proteins and designed ligands, with important implications for drug discovery. Here, we used nuclear magnetic resonance (NMR) spectroscopy to study the dynamics and interactions of the isolated bromodomains, as well as the tandem construct including both domains and the intervening linker, and investigated the effects of binding a tetra-acetylated peptide corresponding to the tail of histone 4. The peptide affinity is lower for both domains in the tandem construct than for the isolated domains. Using 15N spin relaxation, we determined the global rotational correlation times and residue-specific order parameters for BD1 and BD2. Isolated BD1 is monomeric in the apo state but apparently dimerizes upon binding the tetra-acetylated peptide. Isolated BD2 partially dimerizes in both the apo and peptide-bound states. The backbone order parameters reveal marked differences between BD1 and BD2, primarily in the acetyl-lysine binding site where the ZA loop is more flexible in BD2. Peptide binding reduces the order parameters of the ZA loop in BD1 and the ZA and BC loops in BD2. The AB loop, located distally from the binding site, shows variable dynamics that reflect the different dimerization propensities of the domains. These results provide a basis for understanding target recognition by BRD4

Titration of I-BET762 to segmentally labelled BRD4.

<p>(A) ¹H-¹³C SOFAST TROSY HMQC of 12 μM segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C in apo (red) and in complex with 36 μM I-BET762 (blue). Residues undergoing significant changes in chemical shifts upon binding of I-BET762 are labelled. (B) Structure of BRD4(2) (blue envelope, PDB:2YEM) with surface representation of ILV residues colour coded according to whether they have undergone a large (red) small (pink) or no change (white) in chemical shift upon binding of I-BET762. I-BET762 is shown in a bond representation in its expected binding site based on a structural alignment with the structure of the complex BRD2(1):I-BET762 (PDB:2YEK).</p

Methyl-TROSY of segmentally labelled BRD4(1, 2).

<p>(A) ¹H-¹³C SOFAST TROSY HMQC of segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (red). (B) Spectral overlay of (A) with isolated U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (blue). (C) Spectral overlay of (A) with natural abundance BRD4^NL (purple).</p

Overlay of ¹H-¹⁵N TROSY HSQC spectra of uniformly and segmentally labelled BRD4.

<p>(A) ¹H-¹⁵N TROSY HSQC of U-[²H,¹⁵N]-labelled BRD4(1, 2) (black) overlaid with the segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (red). The peaks corresponding to the mutated G339 and G440 are highlighted by a dashed box. (B) Segmentally labelled BRD4(1, 2) with U-[²H,¹⁵N] and ¹³CH₃ ILV methyl labelled BRD4^C (red).</p

SDS-PAGE analysis of Sortase A-mediated ligation.

<p>(A) 36 μM BRD4^C and 18 μM BRD4^NL were reacted using 18 μM SrtA in 50 mM Tris (pH 7.5), 150 mM NaCl and 1 mM TCEP over the course of 21 h. Samples were taken at the timepoints shown and reaction was stopped by addition of SDS running buffer followed by denaturation at 90°C. (B) Illustration of the slow quenching effect by addition of SDS and denaturation at 90°C at timepoints 0 h, 1 h, 2 h and 3 h (left) compared to quenching at 0 h and 3 h timepoints by addition of 20 mM EDTA at 4°C (right).</p