RAS-RAF-MEK-ERK signaling has a well-defined role in cancer biology. Although aberrant pathway activation occurs mostly upstream of the kinase MEK, mutations in MEK are prevalent in some cancer subsets. Here, we found that cancer-related, activating mutations in MEK can be classified into two groups: those that relieve inhibitory interactions with the helix A region and those that are in-frame deletions of the β3-αC loop, which enhance MEK1 homodimerization. The former, helix A–associated mutants, are inhibited by traditional MEK inhibitors. However, we found that the increased homodimerization associated with the loop-deletion mutants promoted intradimer cross-phosphorylation of the activation loop and conferred differential resistance to MEK inhibitors both in vitro and in vivo. MEK1 dimerization was required both for its activation by the kinase RAF and for its catalytic activity toward the kinase ERK. Our findings not only identify a previously unknown group of MEK mutants and provide insight into some key steps in RAF-MEK-ERK activation but also have implications for the design of therapies targeting RAS-ERK signaling in cancers.

Although targeting BRAF mutants with RAF inhibitors has achieved promising outcomes in cancer therapy, drug resistance remains a remarkable challenge, and underlying molecular mechanisms are not fully understood. Here, we characterized a previously unknown group of oncogenic BRAF mutants with in-frame insertions (LLRins506 or VLRins506) of αC-β4 loop. Using structure modeling and molecular dynamics simulation, we found that these insertions formed a large hydrophobic network that stabilizes R-spine and thus triggers the catalytic activity of BRAF. Furthermore, these insertions disrupted BRAF dimer interface and impaired dimerization. Unlike BRAF(V600E), these BRAF mutants with low dimer affinity were strongly resistant to all RAF inhibitors in clinic or clinical trials, which arises from their stabilized R-spines. As predicted by molecular docking, the stabilized R-spines in other BRAF mutants also conferred drug resistance. Together, our data indicated that the stability of R-spine but not dimer affinity determines the RAF inhibitor resistance of oncogenic BRAF mutants.

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent throughout the world and has caused great economic losses to the swine industry. Nonstructural protein 10 (nsp10) is a superfamily 1 helicase participating in multiple processes of virus replication and one of the three most conserved proteins in nidoviruses. Here we report three high resolution crystal structures of highly pathogenic PRRSV nsp10. PRRSV nsp10 has multiple domains, including an N-terminal zinc-binding domain (ZBD), a β-barrel domain, a helicase core with two RecA-like domains, and a C-terminal domain (CTD). The CTD adopts a novel fold and is required for the overall structure and enzymatic activities. Although each domain except the CTD aligns well with its homologs, PRRSV nsp10 adopts an unexpected extended overall structure in crystals and solution. Moreover, structural and functional analyses of PRRSV nsp10 versus its closest homolog, equine arteritis virus nsp10, suggest that DNA binding might induce a profound conformational change of PRRSV nsp10 to exert functions, thus shedding light on the mechanisms of activity regulation of this helicase.In 2006, a large-scale atypical PRRS outbreak caused by the highly pathogenic PRRSV (HP-PRRSV, belonging to PRRSV-2) emerged in China [13,14]. Yan Li and her colleagues demonstrated that nsp9 and nsp10 together contributed to the replication efficiency and the high virulence of HP-PRRSV [15]. Nsp9 contains an RNA-dependent RNA-polymerase (RdRp) domain [16]. Nsp10 belongs to the superfamily 1B (SF1B) Upf1-like family of helicases, which could unwind both DNA and RNA duplexes [17][18][19]. In addition, this family of helicases contains an N-terminal predicted zinc-binding domain (ZBD) which is conserved in all nidovirus helicases, including 12 or 13 conserved Cys and His residues [20]. However, the C-terminal domain (CTD) is variable among nidoviruses. Besides, nsp10 can vary up to approximately 42% on the amino acid level between the two genotypes, and the strains within PRRSV-2 also vary considerably with amino acid differences as high as 8% [21,22]. We previously reported the first structure of the nidovirus helicase, equine arteritis virus (EAV; family Arteriviridae) nsp10, and demonstrated that the CTD perhaps exerts a regulatory function on the helicase core, facilitating coupling between NTPase and polynucleotide binding activities [23]. Another solved structure of nidovirus helicase is Middle East respiratory syndrome coronavirus (MERS-CoV; family Coronaviridae) nsp13 [24]. While the domain organization of nsp13 is similar to EAV nsp10, structural comparisons of the individual domains showed that nsp13 is closely related to Upf1. The N-terminal Cys-His-rich domain (CH domain) of nsp13 is more related to the CH of Upf1 than to the ZBD of EAV nsp10. The helicase core of EAV nsp10 is more compact than that of MERS-CoV nsp13 which has a similar size as Upf1. Meanwhile, MERS-CoV nsp13 does not contain a C-terminal regulatory domain homologous to the CTD of EAV nsp10.Reflecting its ...

Relative of Early Flowing 6 (REF6) is a DNA-sequence-specific H3K27me3/2 demethylase that contains four zinc finger (ZnF) domains and targets several thousand genes in Arabidopsis thaliana. The ZnF domains are essential for binding target genes, but the structural basis remains unclear. Here, we determined crystal structures of the ZnF domains and REF6-DNA complex, revealing a unique REF6-family-specific half-cross-braced ZnF (RCZ) domain and two C2H2-type ZnFs. DNA-binding induces a profound conformational change in the hinge region of REF6. Each REF6 recognizes six bases and DNA methylation reduces the binding affinity. Both the acidic region and basic region are important for the self-association of REF6. The REF6 DNA-binding affinity is determined by the sequence-dependent conformations of DNA and also the cooperativity in different target motifs. The conformational plasticity enables REF6 to function as a global transcriptional regulator that directly binds to many diverse genes, revealing the structural basis for the epigenetic modification recognition.

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