Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.DOI: http://dx.doi.org/10.7554/eLife.24903.001

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.H uman respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children and the immunocompromised. HRSV is a pneumovirus of the Paramyxoviridae family of the order Mononegavirales-the nonsegmented negative-strand RNA viruses. Its genome encodes 10 genes that are each transcribed by an RNA-dependant RNA polymerase (RdRp) into single mRNAs. During transcription, the RdRp uses a single promoter in the 3′ leader region (Le) of the genome (1) and responds to gene start and gene end sequences flanking each gene, directing initiation and termination of mRNA transcription, respectively (2). During genome replication, the RdRp bypasses these signals to synthesize a full-length antigenome. The virus-encoded components needed for RNA replication are the large protein (L), the nucleocapsid protein (N), and the phosphoprotein (P). However, complete transcription of mRNAs also requires the M2-1 transcription antiterminator protein (3, 4).M2-1 prevents premature transcription termination both intra-and intergenically (5, 6). M2-1 is essential for HRSV multiplication although it is not currently known how M2-1 effects its role, and deciphering this role is complicated by its multiple interactions with other viral components, namely P (7, 8), RNA (9), and the matrix protein (M) (10). M2-1 is a 194 amino acid, basic protein that forms a stable tetramer in solution (11). Based on mutational analysis and a partial M2-1 structure determined using NMR (12, 13), M2-1 is predicted to comprise four functionally significant regions: an N-terminal Cys 3 -His 1 zinc-binding domain (ZBD) (14); an alpha-helical region proposed to mediate oligomerization (11); the "core" domain (residues ∼58-177) assigned to RNA-and P-binding; and an unstructured C terminus. The core exh...

The therapeutically relevant hypoxia inducible factor HIF-1α–p300 protein–protein interaction can be orthosterically inhibited with α-helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C-terminal helix of the HIF-1α C-TAD (C-terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side-chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF-1α–p300 PPI inhibitors and the first examples of small-molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF-1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42-residue HIF-1α C-TAD from which it is derived.

The HIF-1α/p300 protein-protein interaction plays a key role in tumor metabolism and thus represents a high value target for anticancer drug-development. Although several studies have identified inhibitor candidates using rationale design, more detailed understanding of the interaction and binding interface is necessary to inform development of superior inhibitors. In this work, we report a detailed biophysical analysis of the native interaction with both peptide and Adhiron phage display experiments to identify novel binding motifs and binding regions of the surface of p300 to inform future inhibitor design.

The state of the art in identifying protein–protein interaction inhibitors of hypoxia inducible factor – a promising target for anticancer drug design – is described.

An extended sequence of α-amino acids in HIF-1α is replaced with a non-natural topographical mimic of an α-helix comprised from an aromatic oligoamide to reproduce its p300 recognition properties.

New routes are developed to allow introduction of sensitive side-chains and synthesis of challenging proteomimetic sequences.

The BCL-2 family is a challenging group of proteins to target selectively due to sequence and structural homologies across the family. Selective ligands for the BCL-2 family regulators of apoptosis are useful as probes to understand cell biology and apoptotic signalling pathways, and as starting points for inhibitor design. We have used phage display to isolate Affimer reagents (non-antibody-binding proteins based on a conserved scaffold) to identify ligands for MCL-1, BCL-x L , BCL-2, BAK and BAX, then used multiple biophysical characterisation methods to probe the interactions. We established that purified Affimers elicit selective recognition of their target BCL-2 protein. For anti-apoptotic targets BCL-x L and MCL-1, competitive inhibition of their canonical protein-protein interactions is demonstrated. Co-crystal structures reveal an unprecedented mode of molecular recognition; where a BH3 helix is normally bound, flexible loops from the Affimer dock into the BH3 binding cleft. Moreover, the Affimers induce a change in the target proteins towards a desirable drug-bound-like conformation. These proof-of-concept studies indicate that Affimers could be used as alternative templates to inspire the design of selective BCL-2 family modulators and more generally other protein-protein interaction inhibitors.