# Molecular dissection of the C-terminal tails of tubulin and the effect of their polyglycylation on binding and microtubule assembly

> **NIH NIH R35** · UNIVERSITY OF COLORADO · 2020 · $360,761

## Abstract

Principal Investigator/Program Director(Last, First, Middle): Hough, Loren E.
 Microtubules (MTs) made from ↵- tubulin heterodimers are important for cell migration, long range transport,
and cell division. A primary site of tubulin regulation is the C-terminal tails (CTTs). Major questions remain
about the molecular mechanism of CTT function and regulation. CTTs affect microtubule length dynamics and
mechanical properties even though they contribute only a small percentage of the binding interface between
adjacent dimers. CTTs are a major site of tubulin post-translational modification (PTM) which regulates tubulin's
binding interactions. PTM of the CTTs alters the processivity of motor proteins1, 2 and the affinity of proteins which
affect MT stability (e.g. MCAK, CLIP-170)3, 4 and proteins which stabilize MTs (e.g. tau).5
 Despite their importance, there are few molecular probes of CTT behavior. Because CTTs are flexible, they
are typically undetectable in electron microscopy or x-ray crystallography studies. NMR is the best approach for
determining the molecular mechanism of CTT regulation of MT polymerization, mechanics, binding and regulation
by PTMs. However, standard techniques for incorporation of heavy isotopes have not worked for tubulin—the
proteins have not yet been made in prokaryotic systems, and tubulin typically down regulates its own expression,
making over-expression difficult in eukaryotic systems. We developed a method to produce heavy isotope labeled
tubulin for study by NMR, allowing for a detailed study of the molecular mechanism of CTT function.
 Building on this breakthrough, we will study the role of CTT polyglycylation in MT regulation. The addition of
glycine residues to glutamate side chains occurs on both tubulin CTTs. First identified as one of the two major
poly-modifications on tubulin, polyglycylation is associated with particularly stable MTs, especially axonemes
in mobile cilia.6 Mutations that decrease polyglycylation are associated with reduced stability of cilia, reduced
numbers of cilia, increases in cell proliferation, and cancer. However, it is not known why polyglycylation causes
these effects. PTMs can operate through a variety of mechanisms: modification of the overall charge distribution,7
alteration of binding interfaces,8 induction of structural changes through allosteric mechanisms,9 and modulation
of disordered protein ensembles.10 We will combine NMR, binding assays, molecular simulation, and cell biology
to test potential mechanisms of CTT function. We will study two focused questions:
1. How does polyglycylation affect MT polymerization dynamics and stiffness? The presence of the CTT
affects MT polymerization dynamics and mechanical properties, suggesting that the CTTs of one dimer interact
with other dimers in the MT lattice.11,12 We will purify heavy isotope-labeled tubulin with varying degrees of
polyglycylation using mutations in TTLL3-family proteins and mutations in the tubulin CTT at polyglycy...

## Key facts

- **NIH application ID:** 10004114
- **Project number:** 5R35GM119755-05
- **Recipient organization:** UNIVERSITY OF COLORADO
- **Principal Investigator:** LOREN E HOUGH
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $360,761
- **Award type:** 5
- **Project period:** 2016-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10004114

## Citation

> US National Institutes of Health, RePORTER application 10004114, Molecular dissection of the C-terminal tails of tubulin and the effect of their polyglycylation on binding and microtubule assembly (5R35GM119755-05). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10004114. Licensed CC0.

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