The Neurospora crassa photoreceptor Vivid tunes blue-light responses and modulates gating of the circadian clock. Crystal structures of dark-state and light-state Vivid reveal a light, oxygen, or voltage Per-Arnt-Sim domain with an unusual N-terminal cap region and a loop insertion that accommodates the flavin cofactor. Photoinduced formation of a cystein-flavin adduct drives flavin protonation to induce an N-terminal conformational change. A cysteine-to-serine substitution remote from the flavin adenine dinucleotide binding site decouples conformational switching from the flavin photocycle and prevents Vivid from sending signals in Neurospora. Key elements of this activation mechanism are conserved by other photosensors such as White Collar-1, ZEITLUPE, ENVOY, and flavin-binding, kelch repeat, F-BOX 1 (FKF1).The PAS (Per-Arnt-Sim) protein superfamily transduces signals from diverse biological cues, often by coupling cofactor chemistry to alterations in protein conformation or association (1). The canonical PAS domain protein photoactive yellow protein (PYP) and the light, oxygen, or voltage (LOV) PAS subclass sense blue light in bacteria, plants, and fungi (2, 3). Despite extensive photochemical and structural characterization of such bluelight sensors (2, 4-8), the mechanism by which cofactor excitation leads to biological signal propagation remains an open question.The filamentous fungus Neurospora crassa employs two blue-light sensors with LOV domains, White Collar-1 (WC-1) and Vivid (VVD) to regulate a variety of light responses (9). WC-1 and nonphotosensitive WC-2 form a complex (WCC) that resets the circadian clock by activating transcription of the clock oscillator protein Frequency (FRQ), as well as many other genes (9, 10). VVD, a small PAS protein devoid of auxiliary domains, tunes Neurospora's blue-light response by attenuating activation of the WCC. VVD is essential for response to changing levels of light and for adaptation under constant light (11)(12)(13)(14). VVD and WC-1 share sequence similarity in a core LOV domain and surrounding regions (15). Swapping the WC-1 core LOV domain with that from VVD maintains some light responses in Neurospora (16). VVD and WC-1 require flavin adenine dinucleotide (FAD) for activity instead of flavin mononucleotide (FMN), which is used by plant and algal LOV-containing proteins known as phototropins (9,12,17,18).We report the crystal structure of VVD in its dark-and light-adapted states and show how chemical changes at the active center generate conformational change at the N terminus of

Blue light regulates many physiological and developmental processes in fungi. Most of the blue light responses in the ascomycete Neurospora crassa are dependent on the two blue light regulatory proteins White Collar (WC)-1 and -2. WC-1 has recently been shown to be the ®rst fungal blue light photoreceptor. In the present study, we characterize the Neurospora protein VIVID. VIVID shows a partial sequence similarity with plant blue light photoreceptors. In addition, we found that VIVID non-covalently binds ā avin chromophore. Upon illumination with blue light, VIVID undergoes a photocycle indicative of the formation of a¯avin-cysteinyl adduct. VVD is localized in the cytoplasm and is only present after light induction. A loss-of-function vvd mutant was insensitive to increases in light intensities. Furthermore, mutational analysis of the photoactive cysteine indicated that the formation of a¯avin-cysteinyl adduct is essential for VIVID functions in vivo. Our results show that VIVID is a second fungal blue light photoreceptor which enables Neurospora to perceive and respond to daily changes in light intensity.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

In Neurospora crassa only two white collar (wc) mutants, wc-1 and wc-2, have been described that seem to be insensitive to light. The pleiotropic phenotypes of these mutants suggest that they represent two central components of blue light signal transduction. The WC proteins have several characteristics of transcription factors consistent with an involvement in transcriptional control of light-regulated genes. Here, we present a biochemical analysis of WC1 and WC2 polypeptides in N. crassa. Using specific antisera against WC1 and WC2, respectively, the subcellular localization of the WC polypeptides was investigated. The WC1 protein was localized exclusively in the nucleus, whereas WC2 was detected in both the nuclear and cytoplasmic fractions. The nuclear localization of WC1 and WC2 was shown to be independent of light and dimerization between the two proteins. In addition, WC1 and WC2 are phosphorylated in response to light. The phosphorylation of WC1 and WC2 was dependent on functional WC1 and WC2 proteins, respectively, which clearly indicated a correlation between the light-dependent phosphorylation and the function of WC1 and WC2 in blue light signaling. However, the light-specific phosphorylation of the WC proteins revealed different kinetics. The phosphorylation of WC1 was transient whereas the WC2 phosphorylation was shown to be stable under constant light conditions. The analysis of the light-dependent phosphorylation of WC1 and WC2 in wc-2 and wc-1 mutants revealed an epistatic relationship for WC1 and WC2 with WC2 acting downstream of WC1 in the signal transduction pathway of blue light.