Cis-nonProline peptides: Genuine occurrences and their functional roles

Abstract

While cis peptides preceding proline can occur about 5% of the time, cis peptides preceding any other residue ("cis-nonPro" peptides) are an extremely rare feature in protein structures, of considerable importance for two opposite reasons. On one hand, their genuine occurrences are mostly found at sites critical to biological function, from the active sites of carbohydrate enzymes to rare adjacent-residue disulfide bonds. On the other hand, a cis-nonPro can easily be misfit into weak or ambiguous electron density, which led to a high incidence of unjustified cis-nonPro over the 2006-2015 decade. This paper uses high-resolution crystallographic data and especially stringent quality-filtering at the residue level to identify genuine occurrences of cis-nonPro and to survey both individual examples and broad patterns of their functionality. We explain the procedure developed to identify genuine cis-nonPro examples with almost no false positives. We then survey a large sample of the varied functional roles and structural contexts of cis-nonPro, including the uses of specific amino acids for particular purposes. We emphasize aspects not previously covered: that cis-nonPro essentially always (except for vicinal disulfides) occurs in well-ordered structure, and especially the great concentration of occurrence in proteins that process or bind carbohydrates (identified by occurrence on the CAZy website).

Keywords: Cys‐cis‐Cys; Gly‐cis‐Gly; Tyr‐cis‐X at active site in chitinases; asp‐cis‐asp; cis‐nonPro overrepresented in carbohydrate‐active enzymes; incorrect cis‐nonPro.

FIGURE 1

Walleye stereo of one of the first cis‐nonPro identified, in dihydrofolate reductase, as shown in the later 1.09 Å resolution 1kms. The cis peptide is marked by a lime trapezoid, while the all‐atom contacts are shown as pairs of dots for van der Waals contacts and darker green pillows for H‐bonds.

FIGURE 2

Six chitinase TIM barrels superimposed, showing the completely conserved cis‐nonPro at the end of β‐strand 8 (all Trp‐cis‐X) and β‐strand 2, and the less conserved ones at the end of β‐strand 4. They are 1goi Serratia marcescens (Kolstad et al., 2002), 1w9p Aspergillus fumigatus (Hurtado‐Guerrero & van Aalten, 2007), 3fy1 acidic mammalian (Olland et al., 2009), 3alf Nicotiana tabacum (Ohnuma et al., 2011), 3n11 Bacillus cereus, missing the cis‐nonPro at strand β4 (Hsieh et al., 2010), and 2uy2 Saccharomyces cerevisiae, missing the cis‐nonPro at strand β4 (Rao et al., 2005), at 1.2–1.7 Å resolution.

FIGURE 3

Walleye stereo of 1fsg, a 1.05 Å phosphoribosyl transferase (PRT) enzyme, showing the many H‐bonds between the cis‐nonPro and the substrate (in pink). Gray balls are Mg ions.

FIGURE 4

ψ1, ϕ2 plots, taken as the most diagnostic two‐dimensional projections from five‐dimensional plots. (a) Includes all cis‐nonPro and shows the very strong preference for extended structure, as well as the X‐cis‐G, G‐cis‐X, and first‐residue alpha that are the only exceptions. (b) Shows the 3 TIM barrel clusters, the G‐cis‐G DHFR and arginase clusters, and the first‐residue alpha clusters discussed in the text.

FIGURE 5

The biologically relevant first‐residue‐alpha cis‐nonPro in 3hhs 1.97 Å prophenyl‐oxidase. The Glu is positioned to make an H‐bond through water (small red sphere) to a ligand of the bi‐nuclear Cu site (large copper‐colored spheres), while 2 other ligands are provided by the second helix. H‐bonds are shown as green pillows of dots.

FIGURE 6

The 2xjp structure of flocculin at 0.85 Å, where both Asps bind the Ca⁺⁺, which binds the mannose (Veelders et al., 2010). Electron density contours are at 1.2 and 3.0σ.

FIGURE 7

The 1wd4 arabinofuranosidase at 2.04 Å resolution (Miyanaga et al., 2004). The vicinal SS bond makes extensive van der Waals contact with undecorated side of the sugar ring, preventing binding of a decorated ring. The atoms of the SS bond are gold spheres, and the N and O of the cis bond are blue and red spheres, both pointed in the same direction. Green dots are close contacts and orange spikes are small overlaps. Electron density contours are at 1.0 and 3.0σ.