# Electron microscopy analysis of novel knob-pocket mechanism critical for intermediate filament assembly

> **NIH NIH R03** · YALE UNIVERSITY · 2020 · $83,750

## Abstract

PROJECT SUMMARY
Intermediate filaments (IFs) are a fundamental fibrous component of the cytoskeleton within cells.
Mutations in IF proteins cause or predispose humans to more than 80 diseases, meaning IFs have an
essential role in human health and disease. Moreover, some IFs have been linked to proliferation and
metastasis of cancers. Examples of IFs include keratins, vimentin, desmin, neurofilaments, and lamins. To
fully correlate genotype with clinical phenotype for IF-based diseases, it is important to understand how
mutations alter the three-dimensional protein structure of IF proteins and the filaments they assemble into.
Currently, the atomic resolution basis for how IF proteins assemble into mature 10-nm IFs is not known
and this represents one of the most critical unmet needs in IF biology. What is known from multiple
biophysical studies is that IFs share a common coiled-coil/helical rod domain that is divided into four helical
regions: denoted helix 1A, 1B, 2A, or 2B. This central rod domain is flanked by variable N-terminal head
and C-terminal tail domains.
This proposal aims to address a deficiency in our understanding of the atomic resolution basis for IF protein
assembly into filaments. In particular, we focus on an anchoring knob-hydrophobic pocket IF assembly
mechanism newly discovered in our lab. This discovery was made from two x-ray crystal structures of
keratin 1/10 helix 1B tetrameric complexes – the IF tetramer is considered the building block for higher-
order filament packing. These structures raised several questions that remain unclear: (1) does the knob-
pocket mechanism regulate the rate and/or the length of IF assembly; (2) is the knob-pocket mechanism
conserved across the six types of IFs; (3) which residues in the knob and pocket are most critical for the
interaction; (4) how do mutants of the knob or pocket alter IF assembly; and (5) can the knob-pocket
mechanism be targeted with peptides or small-molecules to disrupt IF assembly. We believe focusing our
studies on these important questions will advance our mechanistic understanding of IF assembly.
In this project, we examine in depth the biochemical and structural properties of the anchoring knob-
hydrophobic pocket IF assembly mechanism identified our laboratory. In Aim 1 we will use negative-stain
electron microscopy to analyze wild-type and mutant IFs to understand how the loss of the knob-pocket
interaction affects the rate and length of filament formation. Multiple IF systems will be evaluated to
establish the degree of conservation of this mechanism across IFs. In Aim 2 we will selectively mutate
hydrophobic pocket residues to determine which pocket residues are most critical to knob binding. Then,
we will study whether knob peptides can bind to the pocket and prevent IF assembly. Accomplishing these
aims will provide novel insight into how the knob-pocket mechanism governs IF assembly and establish a
foundation for developing targeted therapies of IFs throug...

## Key facts

- **NIH application ID:** 9874543
- **Project number:** 1R03AR076484-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Christopher Gerard Bunick
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $83,750
- **Award type:** 1
- **Project period:** 2020-03-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9874543

## Citation

> US National Institutes of Health, RePORTER application 9874543, Electron microscopy analysis of novel knob-pocket mechanism critical for intermediate filament assembly (1R03AR076484-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9874543. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*