G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in stabilization of GPCR:transducer complexes, together with improvements in cryoelectron microscopy (cryo-EM) have recently been applied to structure-assisted drug design for GPCR agonists. Nonetheless, limitations in the commercial application of these approaches, including the use of nanobody 35 (Nb35) to aid complex stabilization and the high cost of 300 kV imaging, have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated the formation of stable complexes with a modified Gs protein in the absence of Nb35. In parallel, we compare 200 versus 300 kV image acquisition using a Falcon 4 or K3 direct electron detector. Moreover, the 200 kV Glacios-Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

Transient biomolecular interactions play crucial roles in many cellular signaling and regulation processes. However, deciphering the structure of these assemblies is challenging owing to the difficulties in isolating complexes from the individual partners. The additive nature of small-angle X-ray scattering (SAXS) data allows for probing the species present in these mixtures, but decomposition into structural and thermodynamic information is difficult. We present a chemometric approach enabling the decomposition of titration SAXS data into species-specific information. Using extensive synthetic SAXS data, we demonstrate that robust decomposition can be achieved for titrations with a maximum fraction of complex of 0.5 that can be extended to 0.3 when two orthogonal titrations are simultaneously analyzed. The effect of the structural features, titration points, relative concentrations, and noise are thoroughly analyzed. The validation of the strategy with experimental data highlights the power of the approach to provide unique insights into this family of biomolecular assemblies.

To mediate cell-to-cell communication via gap junction channels (GJCs), connexins (Cx) traffic as hexameric hemichannels to the plasma membrane, which dock end-to-end between adjacent cell membranes, thereby forming a dodecameric intercellular conduit. Hemichannels also function independently to mediate the passage of contents between the cytoplasm and extracellular space. To generate hemichannels, the mutation N176Y was introduced into the second extracellular loop of Cx26. The electron cryomicroscopy structure of the hexameric hemichannel in lipid bilayer nanodiscs displays an open pore and a 4-helix bundle transmembrane design that is nearly identical to dodecameric GJCs. In contrast to the high resolution of the transmembrane α-helices, the extracellular loops are less well resolved. The conformational flexibility of the extracellular loops may be essential to facilitate surveillance of hemichannels in apposed cells to identify compatible Cx isoforms that enable intercellular docking. Our results also provide a structural foundation for previous electrophysiologic and permeation studies of Cx hemichannels.

Comment on Structure. 2021 Sep 2;29(9):1003-1013.e4.

The kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

The receptor for advanced glycation end products (RAGE) is an immunoglobulin-type multiligand transmembrane protein expressed in numerous cell types, including the central nervous system cells. RAGE interaction with S100B, released during brain tissue damage, leads to RAGE upregulation and initialization of a spiral proinflammatory associated with different neural disorders. Here, we present the structural characterization of the hetero-oligomeric complex of the full-length RAGE with S100B, obtained by a combination of mass spectrometry-based methods and molecular modeling. We predict that RAGE functions as a tightly packed tetramer exposing a positively charged surface formed by V domains for S100B binding. Based on HDX results we demonstrate an allosteric coupling of the distal extracellular V domains and the transmembrane region, indicating a possible mechanism of signal transmission by RAGE across the membrane. Our model provides an insight into RAGE-ligand interactions, providing a basis for the rational design of the therapeutic modifiers of its activity.

Comment on Structure. 2021 Sep 2;29(9):940-950.e4.

Comment in Structure. 2021 Sep 2;29(9):935-936.

Skp2 and cyclin A are cell-cycle regulators that control the activity of CDK2. Cyclin A acts as an activator and substrate recruitment factor of CDK2, while Skp2 mediates the ubiquitination and subsequent destruction of the CDK inhibitor protein p27. The N terminus of Skp2 can interact directly with cyclin A but is not required for p27 ubiquitination. To gain insight into this poorly understood interaction, we have solved the 3.2 Å X-ray crystal structure of the N terminus of Skp2 bound to cyclin A. The structure reveals a bipartite mode of interaction with two motifs in Skp2 recognizing two discrete surfaces on cyclin A. The uncovered binding mechanism allows for a rationalization of the inhibitory effect of Skp2 on CDK2-cyclin A kinase activity toward the RxL motif containing substrates and raises the possibility that other intermolecular regulators and substrates may use similar non-canonical modes of interaction for cyclin targeting.

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.

Tim chaperones transport membrane proteins to the two mitochondrial membranes. TIM9·10, a 70 kDa protein complex formed by 3 copies of Tim9 and Tim10, guides its clients across the aqueous compartment. The TIM9·10·12 complex is the anchor point at the inner-membrane insertase TIM22. The subunit composition of TIM9·10·12 remains debated. Joint NMR, small-angle X-ray scattering, and MD simulation data allow us to derive a structural model of the TIM9·10·12 assembly, with a 2:3:1 stoichiometry (Tim9:Tim10:Tim12). Both TIM9·10 and TIM9·10·12 hexamers are in a dynamic equilibrium with their constituent subunits, exchanging on a minutes timescale. NMR data establish that the subunits exhibit large conformational dynamics: when the conserved cysteines of the CX3C-Xn-CX3C motifs are formed, short α helices are formed, and these are fully stabilized only upon formation of the mature hexameric chaperone. We propose that the continuous subunit exchange allows mitochondria to control their level of inter-membrane space chaperones.

Comment on Proc Natl Acad Sci U S A. 2020 Oct 13;117(41):25254-25262.

Comment in Structure. 2021 Sep 2;29(9):937-939.

α-Synuclein plays an important role in synaptic functions by interacting with synaptic vesicle membrane, while its oligomers and fibrils are associated with several neurodegenerative diseases. The specific monomer structures that promote its membrane binding and self-association remain elusive due to its transient nature as an intrinsically disordered protein. Here, we use inter-dye distance distributions from bulk time-resolved Förster resonance energy transfer as restraints in discrete molecular dynamics simulations to map the conformational space of the α-synuclein monomer. We further confirm the generated conformational ensemble in orthogonal experiments utilizing far-UV circular dichroism and cross-linking mass spectrometry. Single-molecule protein-induced fluorescence enhancement measurements show that within this conformational ensemble, some of the conformations of α-synuclein are surprisingly stable, exhibiting conformational transitions slower than milliseconds. Our comprehensive analysis of the conformational ensemble reveals essential structural properties and potential conformations that promote its various functions in membrane interaction or oligomer and fibril formation.