SUMMARY RIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the ␣-hydroxy carbons of the substrate (C. . . O Ϸ 3.0 Å), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N. . . O < 3.0 and 2.6 Å, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.T riosephosphate isomerase (TIM) catalyzes the isomerization between dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde 3-phosphate (GAP). The uphill direction from DHAP to GAP is essential for optimal throughput in the glycolytic pathway. To accomplish this reaction, TIM extracts the pro-R hydrogen from the C1 carbon of DHAP and then stereo-specifically introduces a proton to the C2 carbon ( Fig. 1 a and b; ref. 1). Kinetic isotope effects and isotope ''washout'' experiments suggest that the reaction proceeds through a planar cis-enediol or enediolate intermediate of moderate stability (2, 3), and that the energetic landscape is characterized by several steps of competitive timescales (4). The crucial proton transfers between C1 and C2 atoms of the substrate are most likely carried out by a single base, the side chain carboxylate of Glu-165, whereas His-95 and Lys-12 probably facilitate the transfer of the hydroxyl proton from O1 to O2 (Fig. 1a; ref. 5). Structural studies revealed an ␣͞␤ fold, now known as the TIM-barrel (6-8), and elucidated the geometry of the catalytic residues at the active site and their interactions with the ligands (9).TIM is a textbook case in enzymatic enolization chemistry and has become the subject of landmark spectroscopic and computational studies elucidating the details of the mechanism, the protein motions relevant to chemistry, and the design principles that allow efficient and uphill proton transfer in enzyme active sites. Spectroscopic and mutation studies have focused on the polarization of the substrate by catalytic residues as well as on the...

Membrane proteins remain difficult to study by traditional methods. Magic angle spinning solid-state NMR (MAS SSNMR) methods present an important approach for studying membrane proteins of moderate size. Emerging MAS SSNMR methods are based on extensive assignments of the nuclei as a basis for structure determination and characterization of function. These methods have already been used to characterize fibrils and globular proteins and are being increasingly used to study membrane proteins embedded in lipids. This review highlights recent applications to intrinsic membrane proteins and summarizes recent technical advances that will enable these methods to be utilized for more complex membrane protein systems in the near future.

Solid-state NMR 2D spectroscopy was used to correlate carbon backbone and side-chain chemical shifts for uniformly (13)C,(15)N-enriched microcrystalline ubiquitin. High applied field strengths, 800 MHz for protons, moderate proton decoupling fields, 80-100 kHz, and high magic angle sample spinning frequencies, 20 kHz, were used to narrow the most of the carbon line widths to 0.5-0.8 ppm. Homonuclear magnetization transfer was effected by matching the proton RF field to the spinning frequency, the so-called dipolar-assisted rotational resonance (DARR) (Takegoshi, K.; Nakamura, S.; Terao, T. Chem. Phys. Lett. 2001, 344, 631-637), and a mixing time of 20 ms was used to maximize the intensity of one-bond transfers between carbon atoms. This polarization transfer sequence resulted in roughly 14% transfer efficiencies for directly bonded carbon pairs and 4% transfer efficiencies for carbons separated by a third carbon. With this simple procedure, the majority of the one-bond correlations was observed with moderate transfer efficiencies, and many two-bond correlations were also observed with weaker intensities. Spin systems could be identified for more than half of the amino acid side chains, and site-specific assignments were readily possible via comparison with 400 MHz (15)N-(13)C-(13)C correlation spectroscopy (described separately).