SUMMARY RAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and 19F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase.

Key proteins of retroviruses and other RNA viruses are translated and subsequently processed from polyprotein precursors by the viral protease (PR). Processing of the HIV Gag-Pol polyprotein yields the HIV structural proteins and enzymes. Structures of the mature enzymes PR, reverse transcriptase (RT), and integrase (IN) aided understanding of catalysis and design of antiretrovirals, but knowledge of the Pol precursor architecture and function before PR cleavage is limited. We developed a system to produce stable HIV-1 Pol and determined its cryo–electron microscopy structure. RT in Pol has a similar arrangement to the mature RT heterodimer, and its dimerization may draw together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate assembly and maturation of polyprotein precursors.

Many retroviral proteins are initially translated from unspliced full-length RNA as polyprotein precursors that are subsequently processed by the viral protease (PR) to yield the mature forms. In HIV-1, the enzymes, PR, reverse transcriptase (RT), and integrase (IN), are produced as part of the Gag-Pol polyprotein. While structures of the mature proteins have aided our understanding of catalytic mechanisms and the design of antiretroviral drugs, knowledge of the architecture and functional implications of the immature forms prior to PR-mediated cleavage is limited. We developed a system to produce and purify the HIV-1 Pol polyprotein intermediate precursor and determined its high-resolution cryo-EM structure. The RT portion of the polyprotein has an architecture similar to the mature RT p66/p51 heterodimer, and dimerization of the RT portion draws together two PR monomers to activate proteolytic processing. HIV-1 thus may leverage the dimerization interfaces in Pol to regulate the assembly and maturation of the polyprotein precursors.