Insulin signaling controls metabolic homeostasis. Here, we report the cryo-EM structure of full-length insulin receptor (IR) and insulin complex in the active state. This structure unexpectedly reveals that maximally four insulins can bind the ‘T’-shaped IR dimer at four distinct sites related by 2-fold symmetry. Insulins 1 and 1’ bind to sites 1 and 1’, formed by L1 of one IR protomer and α-CT and FnIII-1 of the other. Insulins 2 and 2’ bind to sites 2 and 2’ on FnIII-1 of each protomer. Mutagenesis and cellular assays show that both sites 1 and 2 are required for optimal insulin binding and IR activation. We further identify a homotypic FnIII-2–FnIII-2 interaction in mediating the dimerization of membrane proximal domains in the active IR dimer. Our results indicate that binding of multiple insulins at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer.

SummaryRIG-I and MDA5 sense virus-derived short 5′ppp blunt-ended or long dsRNA, respectively, causing interferon production. Non-signaling LGP2 appears to positively and negatively regulate MDA5 and RIG-I signaling, respectively. Co-crystal structures of chicken (ch) LGP2 with dsRNA display a fully or semi-closed conformation depending on the presence or absence of nucleotide. LGP2 caps blunt, 3′ or 5′ overhang dsRNA ends with 1 bp longer overall footprint than RIG-I. Structures of 1:1 and 2:1 complexes of chMDA5 with short dsRNA reveal head-to-head packing rather than the polar head-to-tail orientation described for long filaments. chLGP2 and chMDA5 make filaments with a similar axial repeat, although less co-operatively for chLGP2. Overall, LGP2 resembles a chimera combining a MDA5-like helicase domain and RIG-I like CTD supporting both stem and end binding. Functionally, RNA binding is required for LGP2-mediated enhancement of MDA5 activation. We propose that LGP2 end-binding may promote nucleation of MDA5 oligomerization on dsRNA.

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3′-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3′ hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5′-end with the foot of the 3′ hairpin and has thus functional implications.