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.

Type 1 insulin-like growth factor receptor (IGF1R) is a receptor tyrosine kinase that regulates cell growth and proliferation, and can be activated by IGF1, IGF2, and insulin. Here, we report the cryo-EM structure of full-length IGF1R–IGF1 complex in the active state. This structure reveals that only one IGF1 molecule binds the Γ-shaped asymmetric IGF1R dimer. The IGF1-binding site is formed by the L1 and CR domains of one IGF1R protomer and the α-CT and FnIII-1 domains of the other. The liganded α-CT forms a rigid beam-like structure with the unliganded α-CT, which hinders the conformational change of the unliganded α-CT required for binding of a second IGF1 molecule. We further identify an L1–FnIII-2 interaction that mediates the dimerization of membrane-proximal domains of IGF1R. This interaction is required for optimal receptor activation. Our study identifies a source of the negative cooperativity in IGF1 binding to IGF1R and reveals the structural basis of IGF1R activation.

Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumor suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small cell lung cancer (NSCLC). Concurrent KRAS mutation and LKB1 loss (KL) specifies aggressive oncological behavior1,2. We show that KL cells and tumors share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate (CP) in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. CPS1 transcription is suppressed by LKB1 via AMPK, and CPS1 expression anticorrelates with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumor growth. Surprisingly, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine/purine ratio, compromises S-phase progression, and induces DNA polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncogenotype imposes a novel metabolic vulnerability related to exquisite dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.