Kinematic Flexibility Analysis: Hydrogen Bonding Patterns Impart a Spatial Hierarchy of Protein Motion

Elastic network models (ENM) and constraint-based, topological rigidity analysis are two distinct, coarse-grained approaches to study conformational flexibility of macromolecules. In the two decades since their introduction, both have contributed significantly to insights into protein molecular mechanisms and function. However, despite a shared purpose of these approaches, the topological nature of rigidity analysis, and thereby the absence of motion modes, has impeded a direct comparison. Here, we present an alternative, kinematic approach to rigidity analysis, which circumvents these drawbacks. We introduce a novel protein hydrogen bond network spectral decomposition, which provides an orthonormal basis for collective motions modulated by non-covalent interactions, analogous to the eigenspectrum of normal modes, and decomposes proteins into rigid clusters identical to those from topological rigidity. Our kinematic flexibility analysis bridges topological rigidity theory and ENM, and enables a detailed analysis of motion modes obtained from both approaches. Our analysis reveals that collectivity of protein motions, reported by the Shannon entropy, is significantly lower for rigidity theory versus normal mode approaches. Strikingly, kinematic flexibility analysis suggests that the hydrogen bonding network encodes a protein-fold specific, spatial hierarchy of motions, which goes nearly undetected in ENM. This hierarchy reveals distinct motion regimes that rationalize protein stiffness changes observed from experiment and molecular dynamics simulations. A formal expression for changes in free energy derived from the spectral decomposition indicates that motions across nearly 40% of modes obey enthalpy-entropy compensation. Taken together, our analysis suggests that hydrogen bond networks have evolved to modulate protein structure and dynamics

Chemical Biology, Biochemical and Structural Studies of MDN1, an AAA Protein Required for Ribosome Biogenesis

Cellular proteins are synthesized by ribosomes, which are ~3 MDa macromolecular complexes comprised of four ribosomal RNAs and ~80 ribosomal proteins in yeast. The biogenesis of such complicated ribonucleoprotein complexes is a highly regulated, multistep process requiring a plethora of more than 200 unique assembly factors. Energy-harnessing enzymes, such as ATPases and GTPases, are needed to remodel the precursors of ribosomes at fast time scales. Mdn1 is an essential dynein-like AAA protein (ATPases Associated with various Activities) that releases specific assembly factors from the precursors of 60S subunit of ribosomes. However, Mdn1’s unusually large size (~5000 amino acids in a single polypeptide) and the transient nature of intermediates of ribosome biogenesis have limited our understanding how Mdn1 remodels pre-60S particles. In addition, the limited homology of Mdn1 to other well-studied proteins, including dyneins, has restricted our understanding of its function. Here, I first combined chemical and biochemical approaches to develop and validate ribozinoindoles (Rbins) as the cell-permeable inhibitors of Mdn1, which are the first potent and selective inhibitors of ribosome biogenesis in eukaryotes. These compounds can be further used to dissect the dynamic functions of Mdn1 during the multistep process of ribosome biogenesis. In addition, I solved three cryo-EM structures of both full-length and truncated Mdn1 (resolution up to 4.0 Å) that provided the first pseudo-atomic models for Mdn1 in two distinct nucleotide states. Remarkably, Mdn1’s the C-terminal MIDAS domain (Metal Ion-Dependent Adhesion Site), which interacts with other ribosome assembly factors, docks onto the N-terminal AAA ring in a nucleotide state-specific manner, even though they are separated by more than 2000 aa. These data suggest that conformational changes in the AAA ring can be directly transmitted to the MIDAS domain, thereby driving the selective release of the MIDAS-bound assembly factors from the precursors of 60S subunit of ribosomes. Together, these chemical biology, biochemical and structural studies of Mdn1 reveal how an AAA protein can contribute to the dynamic ribosome biogenesis process in eukaryotes

Molecular Basis for DNA Repair and RNA Homeostasis Functions of SAMHD1

Sterile Alpha Motif and Histidine-Aspartate domain-containing protein 1 (SAMHD1) is the major negative regulator of dNTP pools in human cells through its unique deoxynucleoside triphosphate triphosphohydrolase activity. This activity constitutes a restriction mechanism against retroviral replication in terminally differentiated cells, as well as a resistance mechanism against nucleoside analogue chemotherapies in some leukemias. SAMHD1 is also one of five gene products associated with Aicardi-Goutieres Syndrome (AGS), an autoimmune disorder characterized by chronic type I interferon signaling. Recent work has shown that SAMHD1-associated AGS pathology arises from its DNA repair functions at double stranded breaks (DSBs), stalled replication forks (RFs), R-loops, and telomeres, as well as a putative function in cellular RNA homeostasis. In the absence of functional SAMHD1, self-derived DNA and RNA structures accumulate and activate the viral nucleic acid sensors cGAS and MDA5, inducing the interferon response associated with AGS. The aim of this dissertation is to determine the molecular basis for these newly discovered functions of SAMHD1. In the first investigation, a mechanism for how CDK2-mediated phosphorylation enables the DNA repair functions of SAMHD1 is established. This modification destabilizes the SAMHD1 tetramer and allows single-stranded DNA (ssDNA) to invade the tetramer interface, inactivating dNTPase activity. These effects are predicted to enable site-directed disassembly of SAMHD1 tetramers at stalled RFs and DSBs. In the second investigation, a comprehensive biochemical characterization of nucleic acid binding by SAMHD1 is reported. This work reveals that SAMHD1 binds specifically to guanine bases in ssDNA and ssRNA by repurposing a GTP binding site. Like GTP binding, guanine base binding to this site allosterically modulates the oligomeric state of SAMHD1, producing dimeric and tetrameric species. TEM micrographs demonstrate that these oligomers form “beads on a string” structures on long RNA molecules that disrupt secondary structure. Implications of these findings with respect to the biological functions of SAMHD1 in DNA repair and RNA homeostasis are discussed

Visualizing cell surface interactions using cryogenic electron microscopy

The study of the three-dimensional structures of biological macromolecules has given us significant insight into life and its mechanisms. Understanding these structures in their native contexts, a challenging but important goal, came closer to reality with the development of electron microscopy. After many years of technological development, we are now starting to understand previously intractable biological phenomena at an unprecedented resolution. One such phenomenon is how neighboring cells interact, both to communicate and send signals, and to adhere and form complex tissue structures. While the molecules that mediate such processes have long been studied in isolation, electron microscopy allows us to examine them in a more native biophysical environment; as hydrated, dynamic molecules tethered to opposed cellular membranes.Imaging unadulterated biological material using electron microscopy requires that the sample be embedded in a thin layer of vitreous ice to immobilize the molecules and protect them from the vacuum of the microscope, and thus is generally referred to as cryogenic electron microscopy (cryo-EM). Samples can be imaged using two common cryo-EM modalities: single particle analysis (SPA), where many two-dimensional projection images of molecules in solution are collected, and cryo-electron tomography (cryo-ET), where the sample is tilted as it is imaged at multiple angles to reconstruct a three-dimensional volume. In this work, I will describe how I have used both SPA and cryo-ET to understand cell surface interactions involving a variety of proteins. The first chapter will look at the cell surface molecules known as the Toll receptors, a family of molecules found in Drosophila melanogaster, with orthologs in mammals known as the Toll-like receptors (TLRs). I will focus on their role in the development of the Drosophila embryo during germ band extension, a kind of convergent extension that is a conserved process through all metazoans. Biophysical assays of the three implicated Toll receptors, Toll-2, -6, and -8, revealed both homophilic and heterophilic interactions. SPA was used to determine the structure of monomeric Toll-2 which closely resembles the overall fold of Toll, whose structure was previously solved by x-ray crystallography. Surface plasmon resonance (SPR) spectroscopy and analytical ultracentrifugation (AUC) showed Toll-6 is a dimer in solution, which I visualized using cryo-EM. The Toll-6 homodimer is a novel dimer interface for Tolls and TLRs, where molecules on the same cell surface have been shown to dimerize in the presence of a wide variety of ligands. In contrast, the Toll-6 dimer is formed in the absence of any ligand and exists in an antiparallel arrangement that could be formed by molecules on opposing cell surfaces. Together, these results provide a biochemical basis for germ band extension which may be further explored through the study of structure-based mutations. While cryo-EM SPA is a powerful tool, cryo-ET allows one to reconstruct three dimensional volumes of highly heterogeneous samples, such as the interior of cells, where molecules of interest may not exist in enough copies to facilitate averaging. This technique, where the sample is imaged multiple times as it is tilted to obtain three-dimensional information of a region of interest, was used to study cell adhesion of a different type: that mediated by the classical cadherins. These calcium-dependent adhesion molecules cluster into adherens junctions, spot-like protein densities found in a wide variety of tissues. In the second chapter, these junctions are recapitulated between synthetic liposome membranes by tethering the adherent cadherin molecules to chemically functionalized lipids. They are then imaged using cryo-ET to reveal higher-order structural details. First, this method is applied to the clustered protocadherins, a family of cadherins that mediate neuronal self-avoidance in mammals. Cryo-ET in combination with x-ray crystallography revealed that clustered protocadherins form extended one-dimensional zippers between membranes, which are a combination of strictly homophilic trans interactions coupled with promiscuous cis interactions. Neurons express unique subsets of the ~50-60 possible isoforms, and when two neuronal processes express identical subsets, which happens only when those processes are a part of the same cell, these linear chains grow and initiate a repulsive signal. If the subsets are different, the chains terminate and no repulsive signal is generated. The same technique has been used previously to study the type I classical cadherins, perhaps the most well-studied members of the cadherin superfamily. In the second half of this chapter, we extend our analysis to include the type II classical cadherins, which possess more complex expression patterns and binding specificities. Cryo-ET of type II cadherin ectodomains tethered to synthetic liposomes revealed that several representative members of this family form only moderately ordered arrays between liposomes, a finding in agreement with their role in cell sorting and migration. However, VE-cadherin, an outlier type II expressed in vascular endothelial cells where it withstands blood pressure, forms extraordinarily ordered junctions. Subtomogram averaging reveals the regularity of this two-dimensional array. In the final chapter, I describe my work on a membrane surface molecule of a different kind, one not involved in cell adhesion but viral infection. The global COVID-19 pandemic gave me the opportunity to contribute to our understanding of SARS-CoV-2 by studying the structure of neutralizing antibodies bound to the viral spike protein, perhaps the most infamous membrane surface protein. The first subchapter describes the initial isolation, neutralization, and structural analysis of antibodies isolated from convalescent COVID-19 patients. This work revealed that patients with severe COVID-19 produce potently neutralizing antibodies that target two spike protein domains: the receptor binding domain (RBD) and the N-terminal domain (NTD). RBD-directed antibodies occlude binding to ACE2, the human receptor that mediates viral fusion, but the neutralization mechanism of NTD-directed antibodies is unknown. The following two subchapters are more detailed structural studies of two specific types of antibodies. The first looks at a class of RBD-directed antibodies derived from the VH1-2 gene, which are some of the most potent and common antibodies against SARS-CoV-2. The heavy chains of these antibodies recognize almost identical epitopes, but the antibodies employ a modular approach to recognize the RBD in either of its possible conformations. The second class are antibodies that target the NTD, which our work revealed all bind to a single antigenic supersite. The final subchapter focuses on emerging SARS-CoV-2 variants and includes the structures of two antibodies that are still capable of neutralizing these new variants. They are also infrequent in the human antibody response to SARS-CoV-2, meaning they put little selective pressure on the virus to produce escape mutations, making them good candidates for monoclonal antibody therapies. Though Drosophila embryogenesis, adherens junction formation, and SARS-CoV-2 neutralization are seemingly unrelated systems, they are united by the incredible flexibility of cryo-EM to visualize biological molecules in more native environments. Whether it is the ability to study multiprotein complexes or assemblies formed between membranes, cryo-EM is a powerful technique that promises to help bridge the divide between structure and function

Characterization and therapeutic exploitation of molecular vulnerabilities in genetically defined lung cancer

Lung cancer is one of the most common cancer types and responsible for the largest number of cancer-related deaths worldwide. Typically, lung cancer arises in individuals with heavy smoking background and only rarely in never-smokers. Various cells of origin within the lung give rise to distinct, molecularly heterogenous lung cancer subtypes with the two major subtypes non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Targeted therapy options also vary significantly between the specific subtypes and while oncogene-driven lung adenocarcinoma (LUAD) is already successfully treated with targeted drugs, no targeted therapies are available in SCLC. LUAD is often driven genetic alterations such as point mutations and rearrangements in genes of receptor tyrosine kinases (RTKs) like EGFR leading to aberrant activation of receptor tyrosine kinase signaling and oncogenic transformation. Mutation-selective small molecule RTK inhibitors have been developed to specifically kill oncogene-addicted cancer cells. Introduction of third generation EGFR inhibitor osimertinib substantially increased survival of EGFR-mutant LUAD patients but on-target resistance mutations such as EGFR G724S limit osimertinib efficacy leading to tumor relapse. Remarkably, we observed that second-generation EGFR inhibitor afatinib displayed selective activity against EGFR G724S in cell line and animal models. In contrast to osimertinib, afatinib still binds to EGFR G724S and reduces cellular viability, EGFR signaling, transformation and in vivo growth of EGFR G724S cells, therefore providing a possible treatment strategy for patients that relapse after osimertinib treatment due to EGFR G724S. Oncogenic gene fusions involving RET also lead to cellular transformation and LUAD tumorigenesis. Previously, multi-kinase inhibitors were used to treat RET-rearranged cancers with limited success due to lack of RET-specificity and RET gatekeeper mutations impeding inhibitor binding. We identified AD80, a type II kinase inhibitor that binds RET in the DFG-out conformation. AD80 displayed selective activity against common RET fusions KIFB-RET and CCDC6-RET and retained activity against RET V804M gatekeeper mutation. AD80 efficiently reduced RET- and downstream signaling as well as RET-associated gene expression. AD80 also displayed in vivo efficacy in CCDC6-RET patient-derived xenograft (PDX) models, demonstrating the potential of type II inhibitors as targeted therapy against RET-rearranged LUAD. In contrast to NSCLC, SCLC is defined by inactivation of tumor suppressors TP53 and RB1 and lacks targetable oncogenic drivers. Frequent activation of MYC transcription factor family members (MYC, MYCL, and MYCN) further accelerate tumor growth and aggressiveness. We found that activation of individual MYC family members entails differential molecular vulnerabilities. MYC overexpression is associated with high levels of DNA damage, repression of BCL2 expression and high apoptotic priming, leading to higher sensitivity towards Aurora kinase and MCL1 inhibition whereas high MYCL/MYCN expression is associated with resistance against these perturbations. Our study highlights that MYC status can be predictive for therapy response and might be used for molecularly-guided, patient stratification for future targeted therapy regimens in SCLC. A rare but very aggressive lung cancer type, NUT carcinoma is driven by BRD4-NUT fusion protein leading to large-scale epigenetic reprogramming and deregulated transcription of genes driving tumorigenesis. Using high-throughput viability screening, we identified that NUT carcinoma cells are preferentially sensitive against CDK9 inhibition. We observed, that CDK9 inhibition increases RNA Polymerase II pausing possibly reverting BRD4-NUT-mediated, transcriptional activation of pro-tumor genes warranting further investigation of CDK9 inhibition in NUT carcinoma

Structural and functional insights into species D adenovirus receptor usage: Implications for oncolytic virotherapy

Adenoviruses are a diverse virus family, infecting a range of animal hosts. The human adenoviruses comprise over 100 types, divided into seven species, A-G. Adenoviruses are clinically important as both engineered therapeutic vectors and pathogens. Adenoviruses are non-membrane bound viruses with double stranded DNA genomes, making them amenable to genetic manipulation and manufacturing at scale. This makes them attractive candidates as therapeutic vectors, currently under development as gene therapy vectors, viral vaccines, and oncolytic viruses. Adenovirus based vectors are in clinical trials for the prevention and treatment of diseases as diverse as Ebola infection and cancer, but are hindered by pre-existing antiviral immunity, leading to neutralisation of the virotherapy before it can have its therapeutic effects, and off-target tissue infections resulting in reduced delivery of therapeutic vector to the site of need. Many natural adenovirus infections do not require medical intervention as they are easily neutralised by the hosts immune system, though they can result in serious disease. Predominantly, this is a concern for immunocompromised patients. However, some types can cause symptoms including gastroenteritis, epidemic keratoconjunctivitis (EKC), and potentially fatal acute respiratory distress syndrome, in healthy adults. Species D adenoviruses are especially diverse, accounting for more than 50% of total adenovirus diversity and many instances of disease, including outbreaks of EKC. They can also be attractive as a basis for therapeutic viruses due to low rates of seroprevelance in the population and favourable immunogenicity profiles. However, little is known about most members of this species or the mechanisms which can make them either pathogenic or therapeutically useful. The work presented herein seeks to better understand how species D adenoviruses infect cells. We therefore investigate their fiber proteins, which mediate primary cell surface receptor binding. We identify previously unknown adenovirus receptor interactions and examine these results in the context of developing new therapeutic adenovirus vectors