Resolving Protein Conformational Plasticity and Substrate Binding Through the Lens of Machine-Learning

A long-standing target in elucidating the biomolecular recognition process is the identification of binding-competent conformations of the receptor protein. However, protein conformational plasticity and the stochastic nature of the recognition processes often preclude the assignment of a specific protein conformation to an individual ligand-bound pose. In particular, we consider multi-microsecond long Molecular dynamics simulation trajectories of ligand recognition process in solvent-inaccessible cavity of two archtypal systems: L99A mutant of T4 Lysozyme and Cytochrome P450. We first show that if the substrate-recognition occurs via long-lived intermediate, the protein conformations can be automatically classified into substrate-bound and unbound state through an unsupervised dimensionality reduction technique. On the contrary, if the recognition process is mediated by selection of transient protein conformation by the ligand, a clear correspondence between protein conformation and binding-competent macrostates can only be established via a combination of supervised machine learning (ML) and unsupervised dimension reduction approach. In such scenario, we demonstrate that a priori random forest based supervised classification of the simulated trajectories recognition process would help characterize key amino-acid residue-pairs of the protein that are deemed sensitive for ligand binding. A subsequent unsupervised dimensional reduction via time-lagged independent component analysis of the selected residue-pairs would delineate a conformational landscape of protein which is able to demarcate ligand-bound pose from the unbound ones. As a key breakthrough, the ML-based protocol would identify distal protein locations which would be allosterically important for ligand binding and characterise their roles in recognition pathways.

A shape-shifting nuclease unravels structured RNA

RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts that may otherwise interfere with cellular programs. The enzyme Dis3-like protein 2 (Dis3L2) is a 3′-5′ exoribonuclease that, through its RNA turnover activity, plays a critical role in human development1. Dis3L2 can independently degrade structured substrates and its targets include many coding and non-coding 3′-uridylated RNAs1-5. While the basis for Dis3L2 substrate recognition has been well-characterized6, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining electron cryo-microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for single-stranded (ss) and double-stranded (ds) RNA processing. We discovered a dramatic conformational change that is triggered by the dsRNA, involving repositioning of two cold shock domains by 70 Å. This movement exposes a trihelix-linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not ssRNA, degradation. These findings reveal the conformational plasticity of this enzyme, and detail a novel mechanism of structured RNA degradation.

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.

The Conformational Plasticity of the Selectivity Filter Methionines Controls the In-Cell Cu(I) Uptake through the CTR1 transporter

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity Copper Transporter 1 (CTR1), being the main in-cell copper (Cu(I)) entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two Methionine triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake-rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import-rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, these outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake.

Asymmetric structures and conformational plasticity of the uncleaved full-length human immunodeficiency virus (HIV-1) envelope glycoprotein trimer

The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41) 3 ] is produced by cleavage of a conformationally flexible gp160 precursor. Gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pre-triggered, “closed” (State-1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more “open” Env conformations (States 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and crosslinking decreased the exposure of pNAb epitopes on cell-surface gp160; however, after detergent solubilization, crosslinked and BMS-806-treated gp160 sampled non-State-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1 N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume State-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a State-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs. IMPORTANCE The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage to a persistent virus like HIV-1.

The structure of the bacterial DNA segregation ATPase filament reveals the conformational plasticity of ParA upon DNA binding

The efficient segregation of replicated genetic material is an essential step for cell division. Bacterial cells use several evolutionarily-distinct genome segregation systems, the most common of which is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of the Vibrio cholerae ParA2 filament bound to DNA, as well as the crystal structures of this protein in various nucleotide states. These structures show that ParA forms a left-handed filament on DNA, stabilized by nucleotide binding, and that ParA undergoes profound structural rearrangements upon DNA binding and filament assembly. Collectively, our data suggest the structural basis for ParA’s cooperative binding to DNA and the formation of high ParA density regions on the nucleoid.

Per aspera ad chaos: a personal journey to the wonderland of intrinsic disorder

This perspective article describes some of the key points of my personal journey through the intriguing world of intrinsically disordered proteins (IDPs). It also shows the evolution of my perception of functional proteins from a standard lock-and-key theory, where a unique function is defined by a unique 3D structure, to the structure–function continuum model, where the structural heterogeneity and conformational plasticity of IDPs define their remarkable multifunctionality and binding promiscuity. These personal accounts of the difficult and lengthy transition from order to disorder paralleled the uneasy and challenging transition in the mind of the scientific community from disbelief in intrinsic disorder to acceptance of IDPs as real entities that play critical biological roles. I hope that this perspective will be of interest to the readers of this journal.