Predicting multibody assembly of proteins

textThis thesis addresses the multi-body assembly (MBA) problem in the context of protein assemblies. [...] In this thesis, we chose the protein assembly domain because accurate and reliable computational modeling, simulation and prediction of such assemblies would clearly accelerate discoveries in understanding of the complexities of metabolic pathways, identifying the molecular basis for normal health and diseases, and in the designing of new drugs and other therapeutics. [...] [We developed] F²Dock (Fast Fourier Docking) which includes a multi-term function which includes both a statistical thermodynamic approximation of molecular free energy as well as several of knowledge-based terms. Parameters of the scoring model were learned based on a large set of positive/negative examples, and when tested on 176 protein complexes of various types, showed excellent accuracy in ranking correct configurations higher (F² Dock ranks the correcti solution as the top ranked one in 22/176 cases, which is better than other unsupervised prediction software on the same benchmark). Most of the protein-protein interaction scoring terms can be expressed as integrals over the occupied volume, boundary, or a set of discrete points (atom locations), of distance dependent decaying kernels. We developed a dynamic adaptive grid (DAG) data structure which computes smooth surface and volumetric representations of a protein complex in O(m log m) time, where m is the number of atoms assuming that the smallest feature size h is [theta](r[subscript max]) where r[subscript max] is the radius of the largest atom; updates in O(log m) time; and uses O(m)memory. We also developed the dynamic packing grids (DPG) data structure which supports quasi-constant time updates (O(log w)) and spherical neighborhood queries (O(log log w)), where w is the word-size in the RAM. DPG and DAG together results in O(k) time approximation of scoring terms where k << m is the size of the contact region between proteins. [...] [W]e consider the symmetric spherical shell assembly case, where multiple copies of identical proteins tile the surface of a sphere. Though this is a restricted subclass of MBA, it is an important one since it would accelerate development of drugs and antibodies to prevent viruses from forming capsids, which have such spherical symmetry in nature. We proved that it is possible to characterize the space of possible symmetric spherical layouts using a small number of representative local arrangements (called tiles), and their global configurations (tiling). We further show that the tilings, and the mapping of proteins to tilings on arbitrary sized shells is parameterized by 3 discrete parameters and 6 continuous degrees of freedom; and the 3 discrete DOF can be restricted to a constant number of cases if the size of the shell is known (in terms of the number of protein n). We also consider the case where a coarse model of the whole complex of proteins are available. We show that even when such coarse models do not show atomic positions, they can be sufficient to identify a general location for each protein and its neighbors, and thereby restricts the configurational space. We developed an iterative refinement search protocol that leverages such multi-resolution structural data to predict accurate high resolution model of protein complexes, and successfully applied the protocol to model gp120, a protein on the spike of HIV and currently the most feasible target for anti-HIV drug design.Computer Science

Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution

Publisher Copyright: © 2022, The Author(s).The origin of viruses remains an open question. While lack of detectable sequence similarity hampers the analysis of distantly related viruses, structural biology investigations of conserved capsid protein structures facilitate the study of distant evolutionary relationships. Here we characterize the lipid-containing ssDNA temperate bacteriophage ΦCjT23, which infects Flavobacterium sp. (Bacteroidetes). We report ΦCjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electron microscopy reveals similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses with a major capsid protein composed of two upright β-sandwiches. The minimalistic structure of ΦCjT23 suggests that this phage serves as a model for the last common ancestor between ssDNA and dsDNA viruses in the Bamfordvirae. Both ΦCjT23 and the related phage FLiP infect Flavobacterium species found in several environments, suggesting that these types of viruses have a global distribution and a shared evolutionary origin. Detailed comparisons to related, more complex viruses not only expand our knowledge about this group of viruses but also provide a rare glimpse into early virus evolution.Peer reviewe

Elucidation of VPS13 and PIKfyve Proteins Functioning in the Regulation of Eukaryotic Lipid Homeostasis

In eukaryotic cells, organelles are surrounded by membranes, which act as barriers to the cytosolic environment. Each subcellular membrane has a distinct lipid composition that is required for its unique organellar function, and is therefore is fundamental for cellular physiology. The unique distributions of organellar lipids result from highly regulated lipid transport networks and the activity of lipid metabolizing enzymes. Most phospholipids are initially synthesized in the ER and transferred to different organelles via vesicular or non-vesicular lipid transport pathways. Lipid transfer proteins (LTPs) localized at membrane contact sites mediate non-vesicular lipid transport. They contain a hydrophobic cavity to solubilize the hydrophobic “tail” of lipids. They either function as “shuttles” that typically ferry a single lipid at a time between membranes, or “bridges” that harbor hydrophobic channels along which more than one lipid can move between membranes at a time. For the first part of my thesis, I investigated the structure and function of VPS13, a novel lipid transfer “bridge” protein, and showed that the protein accommodates a 16nm long hydrophobic lipid transfer channel that mediates bulk lipid transfer. My work marked the identification of the first lipid transfer bridge in eukaryotes and raised several still open questions regarding the molecular mechanism of bridge-like LTPs. I further investigated VPS13’s WD40 domain to provide insights into how VPS13 interacts with membranes at membrane contact sites.The second part of my thesis focused on the modification of phosphatidylinositol (PI), which is essential in signalling. Phosphorylation on different positions of the head group of PI generates several phosphoinositide (PIP) species. Each of them has a unique subcellular localization. PI(3,5)P2 is one of the signature phosphoinositides in endolysosomal membranes, whose level is tightly upregulated in response to stimuli. PI(3,5)P2 is solely synthesized by the PIKfyve lipid kinase and its turnover is catalyzed by the Fig4 lipid phosphatase. Intriguingly, the two proteins, although catalyzing antagonistic reactions, are in the same complex together with a third protein, the scaffold Vac14. Little is known about how the activities of PIKfyve and Fig4 are regulated to prevent futile consumption of ATP. Combining structural and biochemistry studies, I gained insights into the overall architecture of the PIKfyve complex and into the regulatory mechanisms that govern PIKfyve and Fig4 activities

Structural basis of mitochondrial receptor binding and constriction by DRP1.

Mitochondrial inheritance, genome maintenance and metabolic adaptation depend on organelle fission by dynamin-related protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogues mitochondrial dynamics proteins of 49 and 51 kDa (MID49 and MID51) and mitochondrial fission factor (MFF); however, the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryo-electron microscopy structure of full-length human DRP1 co-assembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a marked elongation and rotation of the GTPase domain, bundle-signalling element and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes the polymerization of a linear DRP1 filament with MID49 or MID51. After co-assembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery

Catalytic trajectory of a dimeric nonribosomal peptide synthetase subunit with an inserted epimerase domain.

Nonribosomal peptide synthetases (NRPSs) are modular assembly-line megaenzymes that synthesize diverse metabolites with wide-ranging biological activities. The structural dynamics of synthetic elongation has remained unclear. Here, we present cryo-EM structures of PchE, an NRPS elongation module, in distinct conformations. The domain organization reveals a unique "H"-shaped head-to-tail dimeric architecture. The capture of both aryl and peptidyl carrier protein-tethered substrates and intermediates inside the heterocyclization domain and L-cysteinyl adenylate in the adenylation domain illustrates the catalytic and recognition residues. The multilevel structural transitions guided by the adenylation C-terminal subdomain in combination with the inserted epimerase and the conformational changes of the heterocyclization tunnel are controlled by two residues. Moreover, we visualized the direct structural dynamics of the full catalytic cycle from thiolation to epimerization. This study establishes the catalytic trajectory of PchE and sheds light on the rational re-engineering of domain-inserted dimeric NRPSs for the production of novel pharmaceutical agents

Computational Investigations of Biomolecular Mechanisms in Genomic Replication, Repair and Transcription

High fidelity maintenance of the genome is imperative to ensuring stability and proliferation of cells. The genetic material (DNA) of a cell faces a constant barrage of metabolic and environmental assaults throughout the its lifetime, ultimately leading to DNA damage. Left unchecked, DNA damage can result in genomic instability, inviting a cascade of mutations that initiate cancer and other aging disorders. Thus, a large area of focus has been dedicated to understanding how DNA is damaged, repaired, expressed and replicated. At the heart of these processes lie complex macromolecular dynamics coupled with intricate protein-DNA interactions. Through advanced computational techniques it has become possible to probe these mechanisms at the atomic level, providing a physical basis to describe biomolecular phenomena. To this end, we have performed studies aimed at elucidating the dynamics and interactions intrinsic to the functionality of biomolecules critical to maintaining genomic integrity: modeling the DNA editing mechanism of DNA polymerase III, uncovering the DNA damage recognition/repair mechanism of thymine DNA glycosylase and linking genetic disease to the functional dynamics of the pre-initiation complex transcription machinery. Collectively, our results elucidate the dynamic interplay between proteins and DNA, further broadening our understanding of these complex processes involved with genomic maintenance

Allosteric control of Ubp6 and the proteasome via a bidirectional switch

The interplay of the proteasome and deubiquitinase Ubp6 is crucial for the degradation of ubiquitylated substrates. Here, the authors provide structural insights into the allosteric mechanism by which the activities of both Ubp6 and the proteasome are regulated. The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome