Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation.

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.

The Structure of the Human Respiratory Syncytial Virus M2-1 Protein Bound to the Interaction Domain of the Phosphoprotein P Defines the Orientation of the Complex.

Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause life-threatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90-110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences.IMPORTANCE Human respiratory syncytial virus (HRSV) is a leading cause of respiratory illness, particularly in the young, elderly, and immunocompromised, and has also been linked to the development of asthma. HRSV replication depends on P and L, whereas transcription also requires M2-1. M2-1 interacts with P and RNA at overlapping binding sites; while these interactions are necessary for transcriptional activity, the mechanism of M2-1 action is unclear. To better understand HRSV transcription, we solved the crystal structure of M2-1 in complex with the minimal P interaction domain, revealing molecular details of the M2-1/P interface and defining the orientation of M2-1 within the tripartite complex. The M2-1/P interaction is relatively weak, suggesting high-affinity RNAs may displace M2-1 from the complex, providing the basis for a new model describing the role of M2-1 in transcription. Recently, the small molecules quercetin and cyclopamine have been used to validate M2-1 as a drug target.

The peptide agonist-binding site of the glucagon-like peptide-1 (GLP-1) receptor based on site-directed mutagenesis and knowledge-based modelling.

Glucagon-like peptide-1 (7-36)amide (GLP-1) plays a central role in regulating blood sugar levels and its receptor, GLP-1R, is a target for anti-diabetic agents such as the peptide agonist drugs exenatide and liraglutide. In order to understand the molecular nature of the peptide-receptor interaction, we used site-directed mutagenesis and pharmacological profiling to highlight nine sites as being important for peptide agonist binding and/or activation. Using a knowledge-based approach, we constructed a 3D model of agonist-bound GLP-1R, basing the conformation of the N-terminal region on that of the receptor-bound NMR structure of the related peptide pituitary adenylate cyclase-activating protein (PACAP21). The relative position of the extracellular to the transmembrane (TM) domain, as well as the molecular details of the agonist-binding site itself, were found to be different from the model that was published alongside the crystal structure of the TM domain of the glucagon receptor, but were nevertheless more compatible with published mutagenesis data. Furthermore, the NMR-determined structure of a high-potency cyclic conformationally-constrained 11-residue analogue of GLP-1 was also docked into the receptor-binding site. Despite having a different main chain conformation to that seen in the PACAP21 structure, four conserved residues (equivalent to His-7, Glu-9, Ser-14 and Asp-15 in GLP-1) could be structurally aligned and made similar interactions with the receptor as their equivalents in the GLP-1-docked model, suggesting the basis of a pharmacophore for GLP-1R peptide agonists. In this way, the model not only explains current mutagenesis and molecular pharmacological data but also provides a basis for further experimental design.