Directed Evolution Reveals the Binding Motif Preference of the LC8/DYNLL Hub Protein and Predicts Large Numbers of Novel Binders in the Human Proteome

Abstract

LC8 dynein light chain (DYNLL) is a eukaryotic hub protein that is thought to function as a dimerization engine. Its interacting partners are involved in a wide range of cellular functions. In its dozens of hitherto identified binding partners DYNLL binds to a linear peptide segment. The known segments define a loosely characterized binding motif: [D/S]-4K-3X-2[T/V/I]-1Q0[T/V]1[D/E]2. The motifs are localized in disordered segments of the DYNLL-binding proteins and are often flanked by coiled coil or other potential dimerization domains. Based on a directed evolution approach, here we provide the first quantitative characterization of the binding preference of the DYNLL binding site. We displayed on M13 phage a naïve peptide library with seven fully randomized positions around a fixed, naturally conserved glutamine. The peptides were presented in a bivalent manner fused to a leucine zipper mimicking the natural dimer to dimer binding stoichiometry of DYNLL-partner complexes. The phage-selected consensus sequence V-5S-4R-3G-2T-1Q0T1E2 resembles the natural one, but is extended by an additional N-terminal valine, which increases the affinity of the monomeric peptide twentyfold. Leu-zipper dimerization increases the affinity into the subnanomolar range. By comparing crystal structures of an SRGTQTE-DYNLL and a dimeric VSRGTQTE-DYNLL complex we find that the affinity enhancing valine is accommodated in a binding pocket on DYNLL. Based on the in vitro evolved sequence pattern we predict a large number of novel DYNLL binding partners in the human proteome. Among these EML3, a microtubule-binding protein involved in mitosis contains an exact match of the phage-evolved consensus and binds to DYNLL with nanomolar affinity. These results significantly widen the scope of the human interactome around DYNLL and will certainly shed more light on the biological functions and organizing role of DYNLL in the human and other eukaryotic interactomes.

The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor

“…The final ␤-sheet comprises a total of six strands and is flanked by two ␣-helices (shown in mint, behind the sheet). at the Ϫ3-position, which is often seen in high-affinity LC8 interactors (46), including Nek9 (a kinase that regulates mitotic spindle formation and chromosome separation) (47) and the dynein intermediate chain (DIC; a dynein motor complex component used in cargo recognition) (36) (Fig. 5, A-C).…”

Multiple Recognition Motifs in Nucleoporin Nup159 Provide a Stable and Rigid Nup159-Dyn2 Assembly

“…This binding enhancement involving multiple bivalent dimers is termed "polybivalency" (9, 10) and the IC-light chain assemblage is a canonical example of polybivalency in intrinsically disordered proteins. A common feature among LC8 binding partners, including dynein IC, is that the LC8 recognition motif is disordered in the apo form but is ordered in the LC8-bound form where it packs as a ␤-strand that spans the length of LC8 dimer interface (6,(13)(14)(15)(16)(17).…”

“…Data were acquired on a Bruker Avance 600 MHz spectrometer equipped with a cryogenic 1 H/ 13 C/ 15 N TCI probehead with the z axis gradient coil at 25°C. Five-dimensional HN(CA)CONH and HabCabCONH experiments (29) and an auxiliary three-dimensional HNCO experiment (30) recorded with non-uniform sampling of the indirectly detected dimensions were used for sequential assignments of 500 M 13 C, 15 N-uniformly labeled QT1-6. Interaction of unlabeled Dyn2 with isotopically labeled QT1-6 was characterized by collecting HSQC spectra with Dyn2 at final molar ratios (Nup:Dyn2) of 1:0.8, 1:2, and 1:6.…”

DYNLL2 Dynein Light Chain Binds to an Extended Linear Motif of Myosin 5a Tail That Has Structural Plasticity

“…The width of the binding groove (defined as the distance between 63C to 9C α-carbon atoms of DYNLL (4) Thr and Ile are the most common residues in position 1 of the motifs (9). They are all accommodated into the deepest pocket formed by Y75, F73, F62, L84 and S64 residues of the binding groove ( Figure 8F).…”

“…Unstructured polypeptide regions are often involved in interactions that are mediated by sequential binding sitesknown as linear motifs (7,8) 2 with a conserved glutamine in the arbitrary chosen coreposition 0 (9). A few binding motifs differ considerably from the above consensus sequence; among them is myosin 5a in which position 0 is substituted by a methionine.…”