Polyproteins in structural biology

Abstract

Polyproteins are chains of covalently conjoined smaller proteins that occur in nature as versatile means to organize the proteome of viruses including HIV. During maturation, viral polyproteins are typically cleaved into the constituent proteins with different biological functions by highly specific proteases, and structural analyses at defined stages of this maturation process can provide clues for antiviral intervention strategies. Recombinant polyproteins that use similar mechanisms are emerging as powerful tools for producing hitherto inaccessible protein targets such as the influenza polymerase, for high-resolution structure determination by X-ray crystallography. Conversely, covalent linking of individual protein subunits into single polypeptide chains are exploited to overcome sample preparation bottlenecks. Moreover, synthetic polyproteins provide a promising tool to dissect dynamic folding of polypeptide chains into three-dimensional architectures in single-molecule structure analysis by atomic force microscopy (AFM). The recent use of natural and synthetic polyproteins in structural biology and major achievements are highlighted in this contribution.

Graphical abstract

Figure 1

Natural polyprotein structures. Polyproteins, prevalent in biology, are illustrated here by the structures of the human immune deficiency virus (HIV) immature capsid determined by electron cryo-tomography revealing molecular details of hexameric Gag [24, 26, 27••], the structure of a repeated unit of the ABA-1 nematode polyprotein allergen derived from nuclear magnetic resonance (NMR) spectroscopy [31] and the crystal structure of a single-chain, multi-domain long-chain acyl-CoA carboxylase, LCC [34]. CT stands for carboxyltransferase, BCCP for biotin carboxyl carrier protein, and BC for biotin carboxylase components.

Figure 2

Influenza polymerase. (a) A self-processing recombinant polyprotein was used to express influenza polymerase complex in high quality and quantity in insect cells by using the MultiBac system [37] as illustrated (left). The polymerase was expressed from a single open reading frame encoding for tobacco etch virus NIa protease (TEV), the polymerase subunits PA, PB1 and PB2 and a fluorescent protein (CFP). A second, yellow fluorescent protein (YFP) was inserted elsewhere into the MultiBac baculoviral genome as an expression performance marker. The resulting polyprotein is processed into the individual subunits by highly specific proteolysis mediated by TEV. The polyprotein construct is shown schematically (right) illustrating tag placement and design of spacers in between the subunits (adapted from [6^••]). The C-terminal part of PB1 and the N-terminal part of PB2 co-fold into a helical bundle necessitating careful linker design (boxed). (b) Crystal structure of influenza polymerase bound to cognate viral RNA (vRNA) is shown in a ribbon representation. PA is colored in blue, PB1 in green and PB2 in red. RNA substrate is colored in orange. Cap-binding domain and endonuclease domain are indicated. The structure motif formed by PB1 C-terminal domain and PB2 N-terminal domain is shown in a magnification (right). PB2 N-terminus and PB1 C-terminus are marked.

Figure 3

Linking individual proteins into polypeptide chains. Proteins engineered into single polypeptide chains were used to obtain suitable sample for structure determination of AcrABZ complex [8^••], an alphabody (MA12) to neutralize human interleukin Il-23 (p19 and p40) [13] and the first structure of a histone-modifying enzyme, the Polycomb Repressive Complex (PRC) 1 ubiquitylation module, bound to a nucleosome [12^••]. Linker amino acid segments are marked (L, L1, L2).

Figure 4

SRP pathway revealed by single-chain protein engineering. (a)E. coli signal recognition particle (SRP) protein subunit Ffh (green) and the SRP receptor FtsY (purple) were covalently linked into a single polypeptide chain (left). NG domains are marked. The dashed line indicates the extended polypeptide linker. Bound nucleotide is shown (spheres). The single polypeptide construct is illustrated on the right, detailing the arrangement of FtsY A (blue) and NG (purple) domains, the 30 amino acid glycine/serine rich linker connecting FtsY with Ffh, and the Ffh NG (green) and M (yellow) domains. N and C-termini are marked. (b) Structures of SRP/FtsY bound to a translating ribosome were elucidated by electron cryo-microscopy (color coding as in panel a). 4.5S RNA is colored in orange. EM density and fitted models of the ‘early’ and ‘proofreading’ stages are shown (left, top). Substantial rearrangements are observed as illustrated by the overlay of the EM densities and the model coordinates (left, bottom) (adapted from [10^•]). The cryo-EM structure of the ribosome-SRP-FtsY co-translational targeting complex in the closed state is shown on the right (adapted from [11^•]).

Figure 5

Polyprotein single-molecule structural biology. The setup of atomic force microscopy of polyproteins is shown in a schematic representation (adapted from [14]). The polyprotein is tethered to the gold support resting on the piezoelectronic positioning stage (bottom) on one end, and the tip of a cantilever made of silicon nitride on the other. A laser beam is focused on the back of the cantilever (top). The cantilever is displaced by the force that acts on the polyprotein chain resulting in change of the deflection of the laser beam, recorded by a photodetector. Increasing force causes each domain of the polyprotein to unfold, resulting in characteristic spikes in a force/extension diagram (inset).