Figure 1.
Naïve library design and NL8 structure.
(a) Model of Pyrobaculum aerophilum AspRS, derived from the crystal structure of NL8 and showing the naïve library design. The structure is displayed in blue cartoon representation, with the 17 residues randomised shown as green stick models. (b) Schematic of naïve library design. Residues randomised in the library by NNK codons are shown in red. The annotated secondary structural elements, derived from the crystal structure of NL8, are shown above the sequence as yellow arrows (β-strands; S1–5), pink cylinders (α-helices) and grey wavy lines (loops; L1–4). The GA residues (positions 46 and 47; boxed) are the result of a 6 bp insertion in the wild-type gene immediately following wild-type residue 46, done to facilitate library construction. (c) The NL8 OBody (blue) is shown in complex with HEL (green surface model). Highlighted as stick models are R35 and Y33, interacting with the substrate-binding groove of HEL. (d) The R35 of the NL8 Obody (blue) binds into the HEL active site, in a similar manner to H60 from lysozyme inhibitor protein YkfE from E. coli (pink).
Table 1.
Data collection and refinement statistics for all OBody-HEL complexes reported here.
Figure 2.
Sequences and affinities of OBodies from first round of affinity maturation and AM1L10 structure.
(a) OBody sequences from the first round of affinity maturation, showing only the mutations at the targeted residues and labelled according to their sequence position in the library. For comparison, the equivalent positions from NL8 are also shown. Residues are coloured by polarity or charge (yellows = non-polar, greens = polar, red = acidic, blue = basic). (b) AM1L10 (pale blue Cα trace) in complex with HEL (pale green), superposed on to NL8 (dark blue, r.m.s.d. 0.45 Å) with the NL8-bound HEL also visible (dark green). Substitution A56Y introduces a hydrogen bond with HEL T47 and the subsequent relative shift in HEL position is evident, with a 2.5 Å shift in Cα position at that residue (indicated by red dashed line). (c) AM1L10 (pale blue Cα trace) in complex with HEL (pale green), superposed on to NL8 (dark blue, r.m.s.d. 0.45 Å) with the NL8-bound HEL also visible (dark green). At the top of the binding face, E95 accommodates the shift with a conformational change, maintaining close contact with a lysine and arginine from HEL. Dashed yellow lines are potential hydrogen or electrostatic bonds, labelled with distances in angstroms.
Figure 3.
Phage ELISA, SPR and kinetic data for top hits from third round ofaffinity maturation.
(a) Top hits from the third round of affinity maturation ranked by phage ELISA signal and showing the corresponding L4 sequence. (b) SPR sensorgrams for OBodies AM3L09 (blue line), AM3L15 (orange line) and AM2EP06 (green line) binding to HEL. For visual comparison, sensorgrams are shown only for the highest single concentration of each OBody analysed (AM3L09 at 32 nM, AM3L15 at 128 nM and AM2EP06 at 800 nM). These data were produced on a single chip and are representative of multiple independent analyses, performed with separate chips and protein samples. (c) Kinetic data for each OBody. The AM3L09 and AM3L15 kinetic data were calculated using Graphpad Prism association/dissociation modelling, whereas the affinity of AM2EP06 was calculated using an equilibrium model of maximum response. While the kon for both AM3L15 and AM3L09 are essentially the same, the difference in dissociation constant can be attributed to a substantial decrease (10-fold) in koff. Separate on and off rates could not be determined from the AM2EP06 data.
Figure 4.
Contribution of L4 to binding in five OBody-HEL complexes.
HEL is shown in green as a surface representation in identical orientations in each image and the L4 residues from each OBody variant, coloured according to B-factor relative to the average in each individual case; blue is low B-factor, graduating through green, yellow then red as it increases. The lower-affinity variants NL8 and AM1L10 both show poorly-ordered L4 residues. Note that residues D89, M90, H91 and N92 are missing from the model of AM1L10 L4, as they could not be resolved. Relative stabilisation is evident in L4 of the higher-affinity variants AM3L15 and AM3L09, compared to the parent clone AM2EP06, implying increased involvement in binding. Although the L4 structures of AM3L09 and AM2EP06 have superficially similar configurations, with Y88 and W90 binding in similar positions, W90 L4 from AM3L09 makes a greater number of contacts and is packed more closely. The alpha carbons of N- and C-terminal L4 residues have been labelled with ‘N’ and ‘C’ respectively, to denote the anchor points for the loop in each case.
Figure 5.
Anatomy of the AM3L09-HEL interface.
(a) AM3L09 is coloured blue with interface residues shown as stick models. HEL residues calculated to make a hydrogen bond with AM3L09 are shown as green stick models. Potential hydrogen bonds are indicated by a dashed yellow line. (b) HEL electrostatic surface. The highly electronegative HEL active site (AS) is filled by R35 from AM3L09. (c) AM3L09 electrostatic surface, shown in the same orientation as panel a. The negatively charged patch containing D91 associates with a complementary positively charged patch on the HEL interface. (d) Comparative binding positions of AM3L09 (blue, thick Cα trace) and NL8 (red, narrow Cα trace) to HEL (green surface). (e) The AM3L09-HEL interface, shown in wall-eye stereo. Bridging water molecules between AM3L09 (blue) and HEL (green) are shown as red spheres. Potential hydrogen bonds are indicated by a dashed yellow line labelled with the length in angstroms.
Figure 6.
Differential scanning calorimetry thermal denaturation and HEL inhibition assay for HEL-binding and control OBodies.
(a) Thermal denaturation by differential scanning fluorimetry of HEL-binding OBodies and a control non-HEL-binding OBody U81. Calculated Tm values are shown alongside the number of amino acid mutations as compared to the wild-type AspRS OB-fold domain from P. aerophilum. (b) HEL activity assay showing inhibition by OBodies AM3L09, AM3L15 and AM2EP06 and negative control U81. Lines show the nonlinear fit of a variable-slope dose-response model. Error bars show the 95% confidence interval derived from triplicate data points. The data shown are representative of that obtained from multiple independent repeats of the same assay.
Table 2.
Interface statistics of OBody HEL-binding compared to other known HEL-binding proteins, calculated by PDBePISA.