Retracing the evolution of a modern periplasmic binding protein

Abstract Investigating the evolution of structural features in modern multidomain proteins helps to understand their immense diversity and functional versatility. The class of periplasmic binding proteins (PBPs) offers an opportunity to interrogate one of the main processes driving diversification: the duplication and fusion of protein sequences to generate new architectures. The symmetry of their two‐lobed topology, their mechanism of binding, and the organization of their operon structure led to the hypothesis that PBPs arose through a duplication and fusion event of a single common ancestor. To investigate this claim, we set out to reverse the evolutionary process and recreate the structural equivalent of a single‐lobed progenitor using ribose‐binding protein (RBP) as our model. We found that this modern PBP can be deconstructed into its lobes, producing two proteins that represent possible progenitor halves. The isolated halves of RBP are well folded and monomeric proteins, albeit with a lower thermostability, and do not retain the original binding function. However, the two entities readily form a heterodimer in vitro and in‐cell. The x‐ray structure of the heterodimer closely resembles the parental protein. Moreover, the binding function is fully regained upon formation of the heterodimer with a ligand affinity similar to that observed in the modern RBP. This highlights how a duplication event could have given rise to a stable and functional PBP‐like fold and provides insights into how more complex functional structures can evolve from simpler molecular components.


Supplementary figures and tables
List of Supplementary Figures:

List of Supplementary Tables:
• Table S1.Amino acid sequences of the proteins analyzed in this work.
• Table S2.Obtained values from the SEC-MALS measurements for the different RBP constructs.
• Table S3.DSC thermodynamic parameters (Tm and ΔH) for the different RBP constructs in absence and presence of ribose.
• Table S4.Data collection and refinement statistics for crystal structures.Addition of ribose to the heterodimer appears to stabilise the complex to a degree where it becomes resistant to dissociation in the SDS loading buffer and subsequent heating as indicated by the presence of higher oligomer bands in the presence of ≥1 mM [ribose] (lanes 6-13).Molecular weight has been estimated as indicated by the addition of the molecular weight standard (lane 1 and 14, weights annotated).S2.

Table S2. Obtained values from the SEC-MALS measurements for the different RBP constructs.
± indicates the standard deviation of 3 separate runs.‡ Polydispersity was calculated by Mw/Mn; Mw -weight-average molar mass moment measured by light scattering; Mn -number-average molar mass moment.A ratio Mw/Mn=1 indicates a homogeneous (i.e., monodisperse) sample, because the average mass is independent of the averaging method.Table S4.Data collection and refinement statistics for crystal structures.Statistics for the highest resolution shell are shown in brackets.
S1. Representative HHpred results for the RBP sequence.• Figure S2.Intrinsic fluorescence measurements of the first-and second-generation constructs.• Figure S3.DSC experiments for the first-and second-generation constructs.• Figure S4.SDS-PAGE of RBP, the individual first-generation halves, and the mixed heterodimer.• Figure S5.Crystallographic dimer formed by the asymmetric-unit mate of RBP-N/RBP-Trunc heterodimer crystal structure.• Figure S6.Biophysical characterization of the second-generation constructs.• Figure S7.DSC endotherms for the co-expressed RBP-NN-His/RBP-CN-Strep heterodimer.

Figure S1 .
Figure S1.Representative HHpred results for the RBP sequence.Visualization of the HHpred output showing the query sequence as a black bar.The database matches are shown as red horizontal bars underneath with their respective identifiers.Bar length is indicating its coverage with respect to the query and is colored according to its significance (red as very significant to orange, yellow, green and cyan as less significant).Top and longer bars show the alignment of other full-length PBPs on the query sequence while bottom and shorter bars indicate the alignment of the individual lobes.On the right is the HHpred probability shown for the presented sequence range.Numbering has been adapted to be consistent with uniprot entry Q9X053.

Figure S4 .
Figure S4.SDS-PAGE of RBP, the individual first-generation halves, and the mixed heterodimer.Purified RBP, RBP-N, RBP-Trunc and RBP-N/RBP-Trunc heterodimer (lane 2-5 respectively) show single proteins at the expected molecular weight without major contaminants.Addition of ribose to the heterodimer appears to stabilise the complex to a degree where it becomes resistant to dissociation in the SDS loading buffer and subsequent heating as indicated by the presence of higher oligomer bands in the presence of ≥1 mM [ribose] (lanes 6-13).Molecular weight has been estimated as indicated by the addition of the molecular weight standard (lane 1 and 14, weights annotated).

Figure S5 .
Figure S5.Crystallographic dimer formed by the asymmetric-unit mate of RBP-N/RBP-Trunc heterodimer crystal structure.Each heterodimer is indicated in orange and blue.