The MSKC hosts a number of guest researchers who utilize our facilities for protein production and structural studies. Below, a list of actively underway or concluded projects.
Drosophilia melanogaster juvenile hormone acid O-methytransferase (DmJHAMT)
Drosophilia melanogaster juvenile hormone acid O-methytransferase (DmJHAMT) is an insect enzyme that natively methylates juvenile hormone acids, but also works on fatty acid substrates, such as lauric acid to produce fatty acid methyl esters (FAMEs), a form of biodiesel, both in vitro and in vivo. Production of biodiesel in bacteria using DmJHAMT is comparable to the most currently effective pathway for doing so and uses fewer enzymes, making it a promising candidate for further pathway optimization. Our goal is to express DmJHAMT in a lauric-acid secreting strain of cyanobacteria to produce an organism that can produce biodiesel directly from carbon dioxide.
c-di-GMP Binding Protein PopA
Caulobacter crescentus has been a model system for studying asymmetric cell division. Caulobacterregulates cell cycle events through its master regulator, which is specifically degraded at one cell pole (stalked) during the swarmer-to-stalk (G1-to-S) transition and in the late predivisional cell to enable replication onset and the transcription of over 90 genes. Surprisingly, the degradation adaptor protein for this master regulator, PopA, localizes symmetrically to both cell poles. PopA binds a secondary messenger molecule, c-di-GMP (cdG), which is synthesized only at the stalked pole and degraded only at the opposite pole. Thus, we hypothesize that the differential distribution of cdG suggests that PopA proteins at the two cell poles are intrinsically different. The structures of PopA, with and without, cdG ligand bound will be absolutely critical for understanding PopA’s function and how symmetrically localized proteins contribute to asymmetric cell division.
Images and text courtesy of lead researcher Jiarui Wang.
Photoactive Yellow Protein (PYP)
PYP is a small (~14kDa, 125 residues) cytosolic protein first found in the bacterium Halorhodospira halophila, in which its function has been inferred from UV-vis action spectra to be blue-light phototaxis. PYP has a p-coumaric acid chromophore (pCA), which binds to the protein via a thioester linkage at the cysteine 69 position. Photon absorption by the chromophore can lead to its trans-to-cis isomerization on a picosecond timescale, which initiates a biological signaling pathway and, eventually, phototaxis.
We are studying PYP as a model system in which to examine the biophysical principles associated with rationally designing proteins to perform specific functions. Aided by crystallography, we are investigating the structure-energetics-function relationships in the hydrogen-bonding network around the chromophore in both its electronic ground- and excited-states.
Images and text courtesy of lead researchers Henk Both, Chi-Yun Li and Ben Thompson.
CAPN5 is a calcium-activated regulatory cysteine protease that is expressed throughout the central nervous system and retina. Mutations in the catalytic core of CAPN5 make it hyperactive and cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV; OMIM#193235), a severe, blinding eye disease.
CAPN5 is the first nonsyndromic uveitis gene, and this discovery provides an unprecedented starting point for investigating the substantial gap in our understanding of the molecular basis of ocular inflammation. Structural studies of CAPN5 ADNIV disease mutants will inform how these mutations alter CAPN5 structure and function. Our laboratory is using X-ray crystallography and other biochemical techniques to study the structural mechanisms of CAPN5 activation and to design novel therapeutic inhibitors for ADNIV.
Images and text courtesy of lead researchers Gabriel Velez and Dr. Vinit Mahajan.
Flavin-dependent thymidylate synthase (FDTS)
The Flavin-dependent thymidylate synthase (FDTS) has been recently identified as a unique type of thymidylate synthase enzyme found predominantly in very pathogenic microbes like Mycobacterium tuberculosis, Clostridium difficile, Helicobacter pylori, etc. This enzyme is essential for the survival of these organisms. Thymidylate synthesis is the terminal step in the sole de novo synthetic pathway to deoxythymidine monophosphate (dTMP), a nucleotide essential for the synthesis of DNA. Thymidylate synthase (TS) catalyzes this crucial reaction. TS inhibition stops DNA production, arresting the cell cycle and eventually leading to "thymineless" cell death. The structural and mechanistic differences between the human enzyme and the bacterial enzyme provide an exciting opportunity for drug design. The objective of the project is to develop inhibitors for the FDTS enzymes.
Images and text courtesy of lead researcher Dr. Irimpan Mathews (SSRL/SLAC).
The apicoplast in malarial parasites
Malaria caused by Plasmodium spp parasites has an enormous disease burden that disproportionately affects the world’s poorest and youngest. Our research focuses on elucidating the biology and function of the parasite’s unique plastid organelle, the apicoplast, which is a key anti-malarial drug target. We take innovative approaches to meet the challenges of studying this complex organism, including a chemical rescue that generates “mutant” parasites lacking their apicoplasts! Investigation of Plasmodium biology offers both the potential for important global health impact and an opportunity to explore fascinating eukaryotic biology.
Images and text coutesy of lead researcher and PI Prof. Ellen Yeh.
The 2’3’-cGAMP-STING Pathway
Most current modulators of innate immunity are broad, non-specific, and poorly characterized, due to our lack of understanding of innate immune mechanisms. Dr. Li lab is taking a more targeted approach as they use chemical biology to unveil how innate immunity functions, and in parallel, develop therapeutic hypotheses and drug leads for cancer treatment. Excitingly, immune-modulating small molecules represent an incredibly important and ice-breaking new area for therapeutic development. Reasearch at Li Lab is focused on two immunomodulator molecules: ENPP1 and STING.
Images and text courtesy of lead researchers Prof. Lingyin Li and graduate researchers Jenifer Brown and Sabrina Ergun.
Protein glycosylation is arguably the most manifold posttranslational modification, and aberrant glycosylation is heavily implicated in cancer malignancy. To dissect the roles of glycans in health and disease, we develop chemical tools that allow for the profiling of protein glycosylation sites. To this end, we have engineered a glycosyltransferase to accept a synthetic, unnatural monosaccharide substrate that enables the deconvolution of glycosylation sites.
This project aims to study the structural implications that lead to the extension of glycosyltransferase substrate specificities in molecular detail. This information will be crucial to design optimized substrates that will be used to dissect the biological roles of protein glycosylation.
Images and text courtesy of lead researchers Dr. Carolyn Bertozzi, Dr. Benjamin Schumann and undergraduate researcher Anthony Agbay.
Orphan DNA Methyltransferase
Sequence-specific DNA methylation is a key form of epigenetic modification present in virtually all kingdoms of life. In higher eukaryotes, DNA methylation is associated with gene expression regulation, genomic imprinting, chromatin modification, aging, carcinogenesis and more. In prokaryotes, the overwhelming majority of DNA methyltransferases described to date are part of restriction-modification systems, key elements in bacterial defense against viruses. Recent studies have shown that orphan DNA methyltransferases, which are not associated with any restriction enzyme, are abundant in microbial genomes. However, the function of orphan methyltransferases remains poorly understood. Using structural bioinformatics and comparative genomics we have identified a gene family, widespread in microbial genomes, that encodes for a new type of orphan DNA methyltransferase. The genes that are part of this family are predominantly found in genomic regions that are horizontally transferred and thus, have the potential to easily spread between genomes.
The goal of this project is to characterize the structure and function of this new class of orphan DNA methyltransferases, using genetic and biochemical tools as well as cryo-electron microscopy.
Images and text courtesy of lead researchers Dr. Hila Sberro and Dr. Yana Gofman.
Dynamics of the Microtubule Complex
Microtubule-associated proteins play a critical role in assembling and stabilizing neuronal microtubules. Tau is natively disordered. Its aggregation into paired helical filaments and neurofibrillary tangles is a classical hallmark of a wide range of neurodegenerative diseases commonly known as tauopathies. Recent studies suggest that tau binds at the interface between tubulin heterodimers to modulate the arrangement of individual microtubules into well-organized, evenly spaced bundles. However, the precise structural and dynamic interactions between tau and microtubules are poorly understood.
The objective of this project is to structurally and dynamically characterize the microtubule complex using a hybrid experimental-computational approach. Our expectation is that we will obtain a high-resolution tau-microtubule structure that will allow us to computationally probe its failure and better understand its function. Ultimately, this will allow us to identify common failure mechanisms shared by different types of taupathies including chronic traumatic encephalopathy, Alzheimer’s disease, frontal dementia, and Parkinsonism.
Images and text courtesy of Prof. Ellen Kuhl.
Carbon Cycling Proteins
Carboxylating enoyl-thioester reductases (ECRs) efficiently catalyze the addition of CO2 to the double bond of α,β-unsaturated CoA-thioesters in an NADPH-dependent reaction. As a result, ECRs can “fix” CO2 with an efficiency 100-times greater than ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo). Through our collaboration with the Joint Genome Initiative, Walnut Creek, CA (JGI), we obtained clones of 20 homologs from different bacterial species. Initial crystals have shown diffraction up to 1.9 Å resolution and a complete data set was collected for apo K.setae ECR. Furthermore, this collaboration also resulted in the elucidation of a 2.2 Å structure of a primary ECR (primary defined as directly involved in CO2-fixation); which represent the first model of a primary ECR to be determined using X-ray crystallography.
This project is a pilot collaboration between SLAC and JGI that will form the basis of a potential BER FICUS proposal. BER related themes of the larger proposal will include carbon-fixation (current pilot study with Tobias Erb), lignin processing (Beth Sattely, Stanford) and ammonia-oxidizing archaea (Chris Francis, Stanford).
Images and text courtesy of lead researchers Dr. Hasan DeMirci and Yash Rao.
Multi-drug resistant (MDR) gram negative bacteria are a grave threat to human health worldwide. While these pathogens currently affect a relatively small portion of the global population, the alarming spread of resistance has led officials at the CDC to predict that by 2050, MDR bacteria will claim more lives than cancer.
The combination of a rapidly growing problem and a relatively barren selection of possible solutions is a powerful impetus for the development of novel antibiotics capable of surmounting some of the obstacles faced by traditional small-molecule therapeutics. Protein therapies against gram-negative bacteria are a new approach that may escape common resistance mechanisms by targeting bacterial cells at a receptor necessary for viability, thereby reducing the chances that the bacteria can down-regulate expression as a resistance strategy. However, the thick, double-layered cell wall of gram-negative bacteria make delivery a key hurdle for protein therapies, which are much larger and less cell-permeable than their small-molecule counterparts. Thus, engineering protein antibiotics with increased cell permeability is a vital step towards creating viable biologic therapies for MDR bacterial infections.
The MKSC is also home to an undergraduate led startup, Lyseia, investigating novel biologics for the treatment of multi-drug resistant gram-negative bacteria. Since the beginning of the summer (2016) Lyseia have been carrying out research in the MKSC to obtain proof-of-concept efficacy data for their lead biologics. The team received $10,000 in seed funding from Stanford ChEM-H to pursue this work, following a successful pitch to Stanford faculty, industry professionals, and venture capitalists at the conclusion of the ChEM-H Entrepreneurship Program.
Crystals images and text courtesy of Undergraduate researchers Maria Filsinger Interrante, Zach Rosenthal, and Christian Choe.
Models of Alzheimers disease
There is significant evidence for a central role of inflammation in the development of Alzheimer’s disease (AD). Epidemiological studies indicate that chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD in healthy aging populations. As NSAIDs inhibit the enzymatic activity of the inflammatory cyclooxygenases COX-1 and COX-2, these findings suggest that downstream prostaglandin signaling pathways function in the pre-clinical development of AD.
Current areas of study in the lab focus on identifying cellular and molecular mechanisms of action of downstream prostaglandin pathways using in vitro and in vivo transgenic models of neurological disease.
Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A) is an important mediator of cellular proliferation due in part to its phosphorylation of nuclear factor of activated T-cells (NFAT) transcription factors. DYRK1A has been proposed as a drug target in cancer, down syndrome, and, more recently, Type II Diabetes.
We aim to determine the co-crystal structure of DYRK1A bound to an inhibitor that we discovered through high-content screening. Such a structure would be an invaluable tool to guide our synthetic strategy for increasing potency and selectivity for DYRK1A.
Crystals images and text courtesy of lead researcher Tim Horton and Undergraduate researcher Ben Yeh.
Adeno-associated Viral Receptor
Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood.
We recently identified a protein receptor essential for AAV serotype 2 (AAV2) infection using a genome-wide knockout screen in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor; AAVR). We showed that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently blocked AAV2 infection. Moreover, genetic ablation of AAVR rendered a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR served as a critical host factor for all tested AAV serotypes.
A crystal structure would give crucial insights on where the receptor interacts with the virion and provide guidance for protein engineering to achieve higher AAV transduction efficiencies.
Crystals images and text courtesy of lead researcher Andreas Puschnik of the Carette Lab.
6-Deoxyerthronolide B synthase (DEBS)
DEBS is a prototypical assembly line polyketide synthase (PKS) that catalyzes the formation of the macrocyclic aglycone core of the antibiotic erythromycin. It has presented an intriguing challenge to the fundamental understanding in enzymology of natural product biosynthesis and provided a potentially powerful engineering platform to realize biosynthesis of novel antibiotics.
To develop tools to facilitate structural and dynamic studies of DEBS, monoclonal antibodies have been raised and shown to possess intriguing properties. We have obtained crystals of a couple of DEBS-antibody complexes and we believe that atomic resolution structure models would greatly empower our engineering efforts to further refine these antibody-based tools.
"Most of the things I'm doing these days are completely new to me, and Marc is my main mentor. He'll actually go with me to SLAC and guide me in how to collect my data."
Crystals images and text courtesy of lead researcher Ted Xiuyuan of the Khosla Lab.
The development of effective broad spectrum antiviral therapies remains a highly attractive goal in drug discovery. Antivirals targeting host cell processes have great potential to demonstrate activity against a wide range of viruses, reduce the likelihood of mutational resistance, and serve as frontline therapies for rapidly emerging outbreaks of viral disease such as Zika, Ebola and influenza.
The pyrimidine salvage enzyme uridine-cytidine kinase (UCK2), a ~30kDa that forms a tetramer in its active form, is necessary for uridine salvage, a process which may limit the antiviral activity of DHODH inhibitors in vivo by replenishing cellular ribonucleotide pools. Therefore, inhibition of this target should allow efficient suppression of cellular ribonucleotide levels. Although several molecules have been shown to inhibit UCK2, they are neither efficient in specifically inhibiting the enzyme or available for purchase.
We are aiming to crystallize UCK2 with the identified inhibitors/activators to understand the modulation and the interaction sites, and rationally design better inhibitors. Please note that high-resolution crystal structures of UCK2 was reported before.
Crystals images and text courtesy of lead researcher Ayse Okesli Armlovich of the Khosla Lab.
Split green fluorescent proteins (GFP) with a structural element removed have potentially useful applications in the control of light-driven protein-protein interactions, and in the preparation of semi-synthetic proteins with novel spectroscopic or functional properties. The truncated GFP generated by removing the 11th β -strand has been previously shown to reassemble with synthetic strand 11 peptide only following light activation. The light irradiation is thought to drive a photo-isomerization reaction within the chromophore of the protein, switching the truncated protein from the thermally stable trans-like state to the strand-receptive cis-like state. However, neither the process by which this occurs nor the presumed conformational changes that accompany it are understood to any level of structural detail.
We believe that the atomistic resolution data obtained from X-ray crystallography will be instrumental in solidifying our understanding of the observed phenomena, and perhaps lead to the rational design and engineering of other split fluorescent protein systems with interesting photo-reactive behaviors.
Crystals images and text courtesy of lead researcher Alan Deng of the Boxer Lab.