Sampling the conformational landscapes of transporters and receptors with AlphaFold2

Equilibrium fluctuations and triggered conformational changes often underlie the functional cycles of membrane proteins. For example, transporters mediate the passage of molecules across cell membranes by alternating between inward-facing (IF) and outward-facing (OF) states, while receptors undergo intracellular structural rearrangements that initiate signaling cascades. Although the conformational plasticity of these proteins has historically posed a challenge for traditional de novo protein structure prediction pipelines, the recent success of AlphaFold2 (AF2) in CASP14 culminated in the modeling of a transporter in multiple conformations to high accuracy. Given that AF2 was designed to predict static structures of proteins, it remains unclear if this result represents an underexplored capability to accurately predict multiple conformations and/or structural heterogeneity. Here, we present an approach to drive AF2 to sample alternative conformations of topologically diverse transporters and G-protein coupled receptors (GPCRs) that are absent from the AF2 training set. Whereas models generated using the default AF2 pipeline are conformationally homogeneous and nearly identical to one another, reducing the depth of the input multiple sequence alignments (MSAs) led to the generation of accurate models in multiple conformations. In our benchmark, these conformations were observed to span the range between two experimental structures of interest, suggesting that our protocol allows sampling of the conformational landscape at the energy minimum. Nevertheless, our results also highlight the need for the next generation of deep learning algorithms to be designed to predict ensembles of biophysically relevant states.

Allosteric modulation of the SARS-CoV-2 spike conformation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through binding to angiotensin-converting enzyme 2 (ACE2), which is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural data and real-time analysis of conformational dynamics have shown that S can adopt multiple conformations, which mediate the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Förster resonance energy transfer (smFRET) imaging we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and the B.1 variant (D614G). We found that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicated antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails.

Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel

The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na+ and K+ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NMR to characterize how bound Na+ vs. K+ affects NaK SF structure and dynamics. We find that the extracellular end of the SF is flexible on the ps-ns timescale regardless of bound ion. On a slower timescale, we observe a structural change between the Na+ and K+-bound states, accompanied by increased structural heterogeneity in Na+. We also show direct evidence that the SF structure is communicated to the pore via I88 on the M2 helix. These results support a dynamic SF with multiple conformations involved in non-selective conduction. Our data also demonstrate allosteric coupling between the SF and pore-lining helices in a non-selective cation channel that is analogous to the allosteric coupling previously demonstrated for K+-selective channels, supporting the generality of this model.

Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS

Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' Disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.

Kinematic Modeling and Inverse Kinematics of Serial 6R Fragment of Molecule

Depending on their mobilities around bond axes, molecules (e.g., proteins, DNA, and RNA) can be modeled as robotic manipulators. We focus on the serial 6R fragments, or the fragments containing six revolute joints connected in series, extracted from these molecules. We solved the inverse kinematics problems of the fragments. We obtained multiple conformations that maintained the relative position and orientation between both ends. Raghavan and Roth’s solution effectively conveys all real solutions. However, the solution is not directly applicable when some link lengths are zeros. To cope with the problem, in addition to the known method based on the modified elimination, we introduced the small-length link strategy. Here, by setting sufficiently small values for the zero-length links, we solved the inverse kinematics problems based on Raghavan and Roth’s solution combined with the symbolic formulation. Moreover, we formulated a method to systematically build manipulator models from structural data of molecules. We systematically identified the Danavit-Hartenberg parameters (link length, offset, and twist angle) and joint angles at the conformation in the structural data from the seven pairs of positions of atoms. Finally, using the structural data of a protein stored in the protein data bank, we demonstrated an application example of kinematic modeling and inverse kinematics calculation.

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-like Membrane

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6/m2 to ≃0.1/m2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.

SHAPER: A Web Server for Fast and Accurate SHAPE Reactivity Prediction

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing serves as a convenient and efficient experiment technique for providing information about RNA local flexibility. The local structural information contained in SHAPE reactivity data can be used as constraints in 2D/3D structure predictions. Here, we present SHAPE predictoR (SHAPER), a web server for fast and accurate SHAPE reactivity prediction. The main purpose of the SHAPER web server is to provide a portal that uses experimental SHAPE data to refine 2D/3D RNA structure selection. Input structures for the SHAPER server can be obtained through experimental or computational modeling. The SHAPER server can accept RNA structures with single or multiple conformations, and the predicted SHAPE profile and correlation with experimental SHAPE data (if provided) for each conformation can be freely downloaded through the web portal. The SHAPER web server is available at http://rna.physics.missouri.edu/shaper/.

DO HABITAT NATURAL AO SISTEMA DA ARTE: O CORPO DO ANIMAL COMO EVIDÊNCIA DO COMPLEXO DE MUDANÇAS DO MUNDO / From natural habitat to the art system: the animal’s body as evidence of a changing world

Este artigo situa de que maneira o animal – reconfigurado na forma de objeto de arte – pode ser tomado como evidência do continuum de mudanças que forjaram o mundo. A reprodução “artística” das espécies acompanha o percurso da própria humanidade, e, nos dias de hoje, sua presença estimula novas incumbências para a arte. Obras com essa especificidade têm sido capazes de acionar uma série de problemas de múltiplas conformações, sobretudo, quando os liames de humanos e animais se encontram no foco de instâncias diversificadas. Acompanhando a trajetória desses seres do habitat natural até sua recepção em espaços certificados, vê-se que, no mesmo rumo dos demais artefatos apropriados pelos artistas, trabalhos contendo animais podem ser considerados eminentes para o sistema da arte, a história da arte e a história do mundo.Palavras-chave: Animal na arte; Objetos de arte; História da arte; Arte contemporânea. AbstractThis article situates in what way the animal – reconfigured in the form of an art object – can be taken as evidence of the continuum of changes that forged the world. The “artistic” reproduction of species follows the path of humanity itself and, nowadays, its presence stimulates new tasks for art. Works with this specificity have been able to trigger a series of problems with multiple conformations, especially when the links between humans and animals are the focus of diversified instances. Following the trajectory of these beings from their natural habitat to their reception in certified spaces, one can see that, in the same course as other artifacts appropriated by artists, works containing animals can be considered eminent for the art system, the art history and the history of the world.Keywords: Animal in art; Art objects; Art history; Contemporary art.