User:Fadel A. Samatey/FlgE III/Intrinsically Disordered Flagellar Rod Stretch

This Molecular Tour does not recap all results described in the publication^[1]. It offers visualizations that complement some of those results, as well as some visualizations not described in the paper.

Campylobacter jejuni hook: contacts to ID-Rod-Stretch

The contacts that the ID-Rod-Stretch makes with neighboring chains in the hook assembly are crucial to the stability of the Campylobacter jejuni hook. As detailed in the accompanying paper^[1], mutations in the hook ID-Rod-Stretch caused the flagellum to fail to form, and shortening of the hook ID-Rod-Stretch loop caused flagella to break off at their base. Here we will visualize these crucial stabilizing contacts.

Here is a single monomer protein chain of FlgE from Campylobacter jejuni (FlgE-Cj), colored by domain ():

This monomer is taken from the complete 3.5 Å cryo-EM structure of the hook, so its intrinsically disordered segment (ID-Rod-Stretch) is held in a stable conformation by contacts with adjacent monomers (not shown). .

• .
• Monomer (colored by domain) in the hook assembly. The contacting atoms are colored by chain; that is, each chain is assigned a different color. This reveals that the hook ID-Rod-Stretch is in contact^[7] with four adjacent chains.
• Here, . There are about eleven hydrogen bonds^[7]. There are two salt bridges^[8], and one uncertain cation-pi interaction^[9], both involving Arg58 near the tip of the ID-Rod-Stretch. A little more than a dozen carbon-carbon van der Waals interactions are also present. (Analysis of bond type was done with FirstGlance in Jmol^[10].)

• Here the hook in order to contrast with the next scene: . This reveals that nearly all of the hook contacts to the ID-Rod-Stretch are from Domain 1 in neighboring chains, with a few contacts from a neighboring hook ID-Rod-Stretch and Domain 0 helices. Contacts to hook Domain 0 helices are mostly from neighboring Domain 0 helices, plus 4 atoms from Domain 1 of neighboring chains. It follows that Domain 1 has many contacts from hook ID-Rod-Stretches of neighboring chains. It also has a few from Domain 1 of neighboring chains.

Salmonella enterica hook and rod: ID-Rod-Stretch models

Previous empirical determinations of the structures of the hook and rod of Salmonella enterica did not report structures for the ID-Rod-Stretch^[2][3][4]. Therefore we used the cryo-EM structure of the Campylobacter jejuni hook (including its long hook ID-Rod-Stretch^[6]) as a template to (color key below, same as in Fig. 1d^[6]). The lengths of these ID-Rod-Stretches vary:

In an earlier report^[6], we showed how the Campylobacter hook ID-Rod-Stretch (earlier termed the "L-stretch") extends from the otherwise cylindrical assembly of D0, acting like a "finger" firmly anchoring the hook D1 domains (see L-Stretch Fingers).

Here, we found that shortening the long hook ID-Rod-Stretch of Campylobacter by removing the 21 amino acids at the distal tip of the hook ID-Rod-Stretch, Gln46-Ile66^[12], caused much lower motility, and many of the flagella broke off at the hook^[6].

Salmonella enterica hook: Lys32 is required for assembly

Lys32 (K32) is one of a handful of highly conserved residues in the Salmonella hook ID-Rod-Stretch. The mutant K32A had severely decreased motility. While wild type averaged 12 flagella/cell, less than 10% of K32A cells had any flagella, and then only 1-2/cell. K32A cells produced twice as much mutant FlgE as wild type cells. Individual point mutations at 3 other highly conserved residues (T28, G30, F38) and 2 semi-conserved residues (F31, M41) were created. Each of these five others single-residue mutants had near-normal motility. Thus, K32 is uniquely important among conserved residues in the hook ID-Rod-Stretch.

Here is the ^[13], described above.

This theoretical model suggests why Lys32 is so important. Lys32 (K32) appears to anchor the ID-Rod-Stretch to Domain 1 by forming a salt bridge with highly conserved Glu361 (E361). Lys32 appears also to interact with Asp62 (D62). in ^[1], showing these interactions.

Bear in mind that rotamer positions and distances between residues are very approximate in this homology model. However, similar interactions are seen in the 3.5 Å cryo-EM structure of the Campylobacter hook. There the homologous residues are Lys34 interacting with Gln81 and Glu809 (not shown).

In addition to the interactions shown in Fig. 8A, . The aromatic sidechains might form cation-pi interactions with either Arg95 or Lys32. Residues homologous to Salmonella hook Lys32, Arg95, and Glu361 are highly conserved in bacterial FlgE proteins.

Salmonella enterica hook assembly

A model of the Salmonella enterica hook assembly was constructed using the above-described monomer plus helical symmetry parameters^[16] previously determined by cryo-EM^[17]. This is an approximate model. While the empirical 3.5 Å cryo-EM structure of the Campylobacter hook has a clashscore of 17 (40th percentile)^[11], the Salmonella hook assembly model has a clashscore^[11] of 64 (1st percentile).

Here is a , about half Campylobacter and half Salmonella for direct comparison.

Recall that Campylobacter FlgE has 5 domains (D0-D4), while FlgE of Salmonella is smaller, with only 3 domains (D0-D2). The cryo-EM Campylobacter FlgE has a longer hook ID-Rod-Stretch (57 residues) that curves into an L shape, strengthening the hook for the high speed of rotation of Campylobacter flagella. As mentioned above, mutation of Campylobacter FlgE that shortens the end of the hook ID-Rod-Loop by 21 residues causes most flagella to break off at the hook. .

The hook ID-Rod-Stretch in Salmonella is shorter (37 residues) and, if the homology model is correct, straighter, without the pronounced L shape seen in Campylobacter. These differences may be seen more clearly when .