Programmed Protein Self-Assembly Driven by Genetically Encoded Intein-Mediated Native Chemical Ligation

Harnessing and controlling self-assembly is an important step in developing proteins as novel biomaterials. With this goal, here we report the design of a general genetically programmed system that covalently concatenates multiple distinct protein domains into specific assembled arrays. It is driven by iterative intein-mediated native chemical ligation (NCL) under mild native conditions. The system uses a series of initially inert recombinant protein fusions that sandwich the protein modules to be ligated between one of a number of different affinity tags and an intein protein domain. Orthogonal activation at opposite termini of compatible protein fusions, via protease and intein cleavage, coupled with sequential mixing directs an irreversible and traceless stepwise assembly process. This gives total control over the composition and arrangement of component proteins within the final product, enabled the limits of the systemreaction efficiency and yieldto be investigated, and led to the production of “functional” assemblies

Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch.C.M. is supported by a BBSRC studentship. B.B.S.R.C Grant E005187/1 and QMUL supported this work and J.J.PThis is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jmb.2016.02.02

Exploring the "N-Terminal Anchor" Binding Interface of the T3SS Chaperone-Translocator Complexes from P. aeruginosa.

The type III secretion system is a large multiprotein complex that many Gram-negative bacteria use for infection. A crucial part of the complex is its translocon pore formed by two proteins: the major and minor translocators. The pore completes a proteinaceous channel from the bacterial cytosol through the host cell membrane and allows the direct injection of bacterial toxins. Effective pore formation is predicated by the translocator proteins binding to a small chaperone within the bacterial cytoplasm. Given the vital role of the chaperone-translocator interaction, we investigated the specificity of the "N-terminal anchor" binding interface present in both translocator-chaperone complexes from Pseudomonas aeruginosa. Isothermal calorimetry (ITC), alanine scanning, and the selection of a motif-based peptide library using ribosome display were used to characterize the major (PopB) and minor (PopD) translocator interactions with their chaperone PcrH. We show that 10 mer PopB51-60 and PopD47-56 peptides bind to PcrH with a KD of 148 ± 18 and 91 ± 9 μM, respectively. Moreover, mutation to alanine of each of the consensus residues (xxVxLxxPxx) of the PopB peptide severely affected or completely abrogated binding to PcrH. When the directed peptide library (X-X-hydrophobic-X-L-X-X-P-X-X) was panned against PcrH, there was no obvious convergence at the varied residues. The PopB/PopD wild-type (WT) sequences were also not prevalent. However, a consensus peptide was shown to bind to PcrH with micromolar affinity. Thus, selected sequences were binding with similar affinities to WT PopB/PopD peptides. These results demonstrate that only the conserved "xxLxxP" motif drives binding at this interface

Computational design of self-assembling cyclic protein homo-oligomers

Self-assembling cyclic protein homo-oligomers play important roles in biology, and the ability to generate custom homo-oligomeric structures could enable new approaches to probe biological function. Here we report a general approach to design cyclic homo-oligomers that employs a new residue-pair-transform method to assess the designability of a protein-protein interface. This method is sufficiently rapid to enable the systematic enumeration of cyclically docked arrangements of a monomer followed by sequence design of the newly formed interfaces. We use this method to design interfaces onto idealized repeat proteins that direct their assembly into complexes that possess cyclic symmetry. Of 96 designs that were characterized experimentally, 21 were found to form stable monodisperse homo-oligomers in solution, and 15 (four homodimers, six homotrimers, six homotetramers and one homopentamer) had solution small-angle X-ray scattering data consistent with the design models. X-ray crystal structures were obtained for five of the designs and each is very close to their corresponding computational model

Exploring the 'N-terminal arm' & 'Convex surface' Binding Interfaces of the T3SS Chaperone-Translocator Complexes from P. Aeruginosa

One infection method widely used by many gram-negative bacteria involves a protein nanomachine called the Type Three Secretion System (T3SS). The T3SS enables the transportation of bacterial “toxins” via a proteinaceous channel that directly links the cytosol of the bacteria and host cell. The channel from the bacteria is completed by a translocon pore formed by two proteins named the major and minor translocators. Prior to pore formation, the translocator proteins are bound to a small chaperone within the bacterial cytoplasm. This interaction is crucial to effective secretion. Here we investigated the specificity of the binding interfaces of the translocator–chaperone complexes from Pseudomonas aeruginosa via the selection of peptide and protein libraries based on its chaperone PcrH. Five libraries encompassing PcrH’s N-terminal and central α-helices were panned, using ribosome display, against both the major (PopB) and minor (PopD) translocator. Both translocators were shown to significantly enrich a similar pattern of WT and non-WT sequences from the libraries. This highlighted key similarities/differences between the interactions of the major and minor translocators with their chaperone. Moreover, as the enriched non-WT sequences were specific to each translocator, it would suggest that PcrH can be adapted to bind each translocator individually. The ability to evolve such proteins indicates that these molecules may provide promising anti-bacterial candidates

Sequence analyses reveal that a TPR–DP module, surrounded by recombinable flanking introns, could be at the origin of eukaryotic Hop and Hip TPR–DP domains and prokaryotic GerD proteins

The co-chaperone Hop [heat shock protein (HSP) organising protein] is known to bind both Hsp70 and Hsp90. Hop comprises three repeats of a tetratricopeptide repeat (TPR) domain, each consisting of three TPR motifs. The first and last TPR domains are followed by a domain containing several dipeptide (DP) repeats called the DP domain. These analyses suggest that the hop genes result from successive recombination events of an ancestral TPR–DP module. From a hydrophobic cluster analysis of homologous Hop protein sequences derived from gene families, we can postulate that shifts in the open reading frames are at the origin of the present sequences. Moreover, these shifts can be related to the presence or absence of biological function. We propose to extend the family of Hop co-chaperons into the kingdom of bacteria, as several structurally related genes have been identified by hydrophobic cluster analysis. We also provide evidence of common structural characteristics between hop and hip genes, suggesting a shared precursor of ancestral TPR–DP domains