Key Interaction Modes of Dynamic +TIP Networks

Abstract

Dynamic microtubule plus-end tracking protein (+TIP) networks are implicated in all functions of microtubules, but the molecular determinants of their interactions are largely unknown. Here, we have explored key binding modes of +TIPs by analyzing the interactions between selected CAP-Gly, EB-like, and carboxy-terminal EEY/F-COO(-) sequence motifs. X-ray crystallography and biophysical binding studies demonstrate that the beta2-beta3 loop of CAP-Gly domains determines EB-like motif binding specificity. They further show how CAP-Gly domains serve as recognition domains for EEY/F-COO(-) motifs, which represent characteristic and functionally important sequence elements in EB, CLIP-170, and alpha-tubulin. Our findings provide a molecular basis for understanding the modular interaction modes between alpha-tubulin, CLIPs, EB proteins, and the dynactin-dynein motor complex and suggest that multiple low-affinity binding sites in different combinations control dynamic +TIP networks at microtubule ends. They further offer insights into the structural consequences of genetic CAP-Gly domain defects found in severe human disorders.

Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy

“…At the same time, as shown in Fig. 3D, loop regions in CAP-Gly underwent pronounced conformational changes upon the formation of a complex with MTs, compared with the free CAP-Gly and CAP-Gly/EB1 complex (15,23,26). These changes resulted in sidechains adopting different orientations in free and MTand EB1-bound states (Fig.…”

“…Fig. 1 illustrates a cosedimentation assay of a typical MAS NMR sample of the U-13 C, 15 N-CAP-Gly/MT complex; CAP-Gly-MT binding assays performed under different conditions are also presented in SI Appendix. The results are fully consistent with our prior findings (22), indicating that CAP-Gly/MT binding is specific and that the binding affinity is estimated in the micromolar range.…”

“…All spectra were recorded at 20.0 T; the CORD mixing time was 500 ms. Intermolecular Interface of CAP-Gly with Microtubules. Determination of intermolecular interfaces formed by a biomolecule interacting with its binding partner by MAS NMR generally relies on (i) analysis of chemical shift perturbations or intensity changes observed for the biomolecule in the complex with respect to the free biomolecule (31,32) and (ii) analysis of intermolecular dipolar-based heteronuclear correlations in differentially isotopically enriched samples, where one biomolecule contains a certain set of isotope labels (e.g., 13 C) and its binding partner a different kind of labels (e.g., 15 N) (33)(34)(35). For analysis of the CAP-Gly/MT interface, we could not use either approach because (i) the chemical shift perturbations are relatively small and present throughout the entire protein and (ii) isotopic labeling of MTs is a challenge.…”

“…Although impressive advances have been made using X-ray diffraction, electron microscopy, biochemistry, and cell biology methods (7,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), including the recent 4.0-Å cryo-EM structure of the dynactin complex (21), essential information concerning the mechanisms of microtubule-based transport is lacking. There are no atomic-resolution structures of motor proteins assembled on polymerized microtubules.…”

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

“…The critical dynactin subunit for plus‐end tracking is p150 Glued (called p150 here). Its N‐terminal CAP‐Gly domain protrudes from the shoulder of the dynactin complex (Chowdhury et al , 2015; Urnavicius et al , 2015) and binds directly to microtubules (Culver‐Hanlon et al , 2006; Peris et al , 1998; Ross et al , 2006), as well as to EBs (Honnappa et al , 2006). In vitro reconstitution experiments with a p150 fragment showed that the p150 CAP‐Gly domain and the first coiled‐coil of p150, which interacts with the intermediate chain of the dynein complex (Karki & Holzbaur, 1995; Vaughan & Vallee, 1995; King et al , 2003), were sufficient for mediating EB‐dependent end tracking of the human dynein complex (Duellberg et al , 2014).…”