Papers by Jeremiah Farelli
Proceedings of the National Academy of Sciences of the United States of America, Jan 6, 2015
Large-scale activity profiling of enzyme superfamilies provides information about cellular functi... more Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution o...
ABSTRACT Since the first genomes were sequenced, there has been an exponential increase of protei... more ABSTRACT Since the first genomes were sequenced, there has been an exponential increase of protein sequences deposited into databases worldwide. This increase in sequence data has allowed for dramatic improvements in our understanding of the metabolism of organisms. Unfortunately, as the number of protein sequences grows, the number of definitive functional assignments diminishes. Current methods involve comparison of sequence identity between known proteins and newly sequenced ones. As the sequence identity decreases, proteins are annotated as "hypothetical," and certainty in annotation shrinks. Thus a strategy for reliably determining protein function is necessary. The HADSF consists of Mg2+-dependent enzymes that catalyze a wide range of reactions including dehalogenation, phosphoryltransfer and dephosphorylation. Here we present the role that high-throughput screening (HTS) might play in functional discovery for families with members having common chemical function but divergence in physiological substrate (e.g., kinases). We highlight five areas of discovery enabled by the HTS-guided substrate profiling: 1) uncovering new metabolic pathways, 2) tracking of orthologs, 3) distribution of promiscuous versus specific family members, 4) annotation of hypothetical proteins, 5) functional assignment of proteins with known structures. This research is supported by the NIGMS (U54 GM093342).
Enzyme function prediction remains an important open problem. Though structure-based modeling, su... more Enzyme function prediction remains an important open problem. Though structure-based modeling, such as metabolite docking, can identify substrates of some enzymes, it is ill-suited to reactions that progress through a covalent intermediate. Here we investigated the ability of covalent docking to identify substrates that pass through such a covalent intermediate, focusing particularly on the haloalkanoate dehalogenase superfamily. In retrospective assessments, covalent docking recapitulated substrate binding modes of known cocrystal structures and identified experimental substrates from a set of putative phosphorylated metabolites. In comparison, noncovalent docking of high-energy intermediates yielded nonproductive poses. In prospective predictions against seven enzymes, a substrate was identified for five. For one of those cases, a covalent docking prediction, confirmed by empirical screening, and combined with genomic context analysis, suggested the identity of the enzyme that catalyzes the orphan phosphatase reaction in the riboflavin biosynthetic pathway of Bacteroides.
Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, functio... more Lanthanide-binding tags (LBTs) are valuable tools for investigation of protein structure, function, and dynamics by NMR spectroscopy, X-ray crystallography, and luminescence studies. We have inserted LBTs into three different loop positions (denoted L, R, and S) of the model protein interleukin-1β (IL1β) and varied the length of the spacer between the LBT and the protein (denoted 1-3). Luminescence studies demonstrate that all nine constructs bind Tb 3þ tightly in the low nanomolar range. No significant change in the fusion protein occurs from insertion of the LBT, as shown by two X-ray crystallographic structures of the IL1β-S1 and IL1β-L3 constructs and for the remaining constructs by comparing the 1 H-15 N heteronuclear single-quantum coherence NMR spectra with that of the wild-type IL1β. Additionally, binding of LBT-loop IL1β proteins to their native binding partner in vitro remains unaltered. X-ray crystallographic phasing was successful using only the signal from the bound lanthanide. Large residual dipolar couplings (RDCs) could be determined by NMR spectroscopy for all LBT-loop constructs and revealed that the LBT-2 series were rigidly incorporated into the interleukin-1β structure. The paramagnetic NMR spectra of loop-LBT mutant IL1β-R2 were assigned and the Δχ tensor components were calculated on the basis of RDCs and pseudocontact shifts. A structural model of the IL1β-R2 construct was calculated using the paramagnetic restraints. The current data provide support that encodable LBTs serve as versatile biophysical tags when inserted into loop regions of proteins of known structure or predicted via homology modeling.
Structure (London, England : 1993), Jan 6, 2013
A Type 4b secretion system (T4bSS) is required for Legionella growth in alveolar macrophages. Icm... more A Type 4b secretion system (T4bSS) is required for Legionella growth in alveolar macrophages. IcmQ associates with IcmR, binds to membranes, and has a critical role in the T4bSS. We have now solved a crystal structure of IcmR-IcmQ to further our understanding of this complex. This structure revealed an amphipathic four-helix bundle, formed by IcmR and the N-terminal domain of IcmQ, which is linked to a novel C-terminal domain of IcmQ (Qc) by a linker helix. The Qc domain has structural homology with ADP ribosyltransferase domains in certain bacterial toxins and binds NAD(+) with a dissociation constant in the physiological range. Structural homology and molecular dynamics were used to identify an extended NAD(+) binding site on Qc, and the resulting model was tested by mutagenesis and binding assays. Based on the data, we suggest that IcmR-IcmQ binds to membranes, where it may interact with, or perhaps modify, a protein in the T4bSS when NAD(+) is bound.
PLoS pathogens, 2014
Parasitic nematodes are responsible for devastating illnesses that plague many of the world's... more Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhi...
Biochemistry, Jan 29, 2014
Herein, the structural determinants for substrate recognition and catalysis in two hotdog-fold th... more Herein, the structural determinants for substrate recognition and catalysis in two hotdog-fold thioesterase paralogs, YbdB and YdiI from Escherichia coli, are identified and analyzed to provide insight into the evolution of biological function in the hotdog-fold enzyme superfamily. The X-ray crystal structures of YbdB and YdiI, in complex with inert substrate analogs, determined in this study revealed the locations of the respective thioester substrate binding sites and the identity of the residues positioned for substrate binding and catalysis. The importance of each of these residues was assessed through amino acid replacements followed by steady-state kinetic analyses of the corresponding site-directed mutants. Transient kinetic and solvent (18)O-labeling studies were then carried out to provide insight into the role of Glu63 posited to function as the nucleophile or general base in catalysis. Finally, the structure-function-mechanism profiles of the two paralogs, along with that...
The Journal of biological chemistry, Jan 31, 2014
Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal ... more Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal to the successful acquisition of novel biological functions. Action on multiple substrates (substrate ambiguity) can be harnessed for performance of functions in the cell that supersede catalysis of a single metabolite. These functions include proofreading, scavenging of nutrients, removal of antimetabolites, balancing of metabolite pools, and establishing system redundancy. In this review, we present examples of enzymes that perform these cellular roles by leveraging substrate ambiguity and then present the structural features that support both specificity and ambiguity. We focus on the phosphatases of the haloalkanoate dehalogenase superfamily and the thioesterases of the hotdog fold superfamily.
Structure, 2009
During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells an... more During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells and this process requires a Type IVb secretion system (T4bSS). IcmQ is a critical component of this translocase and associates with IcmR. In this report, we show that the N-terminal domain of IcmQ (Qn) mediates IcmQ dimerization, while the C-terminal domain with a linker region promotes a stable membrane association of IcmQ. We then determined crystal structures of Qn with the interacting domain of IcmR. In this complex, each protein forms an α-helical hairpin within a parallel 4-helix bundle. We find that IcmR binding to IcmQ prevents dimerization of IcmQ and blocks membrane permeabilization. However, IcmR does not completely block membrane association of IcmQ. The amphipathic nature of Qn suggests two models for how IcmQ may permeabilize membranes. The Rm-Qn structure also suggests how hyper-variable IcmR-like proteins in other Legionellae may interact with their IcmQ partners to regulate IcmQ function.
Protein Science, 2012
Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system fo... more Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solventexposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (a-helix to p-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.
Journal of Molecular Biology, 2011
Journal of Molecular Biology, 2011
Villin headpiece is a small autonomously folding protein that has emerged as a model system for u... more Villin headpiece is a small autonomously folding protein that has emerged as a model system for understanding the fundamental tenets governing protein folding. In this communication, we employ NMR and X-ray crystallography to characterize a point mutant, H41F, which retains actinbinding activity, is more thermostable but, interestingly, does not exhibit the partially folded intermediate observed of either wild-type or other similar point mutants.
Biochemistry, 2011
The explosion of protein sequence information requires that current strategies for function assig... more The explosion of protein sequence information requires that current strategies for function assignment must evolve to complement experimental approaches with computationally-based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown Haloalkanoate Dehalogenase superfamily member BT2127 (Uniprot accession # Q8A5V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics/structure/mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with k cat /K m value for pyrophosphate of ∼1 × 10 5 M −1 s −1 ), together with the gene context, supports the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of the wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure guided site directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domaindomain association. Based on this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172 and Glu47 were used as + This work was supported by NIH grants U54 GM093342 (K.N.A., D.D.-M., and S. A.), N.I.H. grant GM61099 * To whom correspondence should be addressed regarding X-ray structure determination (S.A. or K.N.A.) or kinetic analyses (
Biochemistry, 2012
The mammalian brown fat inducible thioesterase variant 2 (BFIT2), also known as ACOT11, is a mult... more The mammalian brown fat inducible thioesterase variant 2 (BFIT2), also known as ACOT11, is a multimodular protein containing two consecutive hotdog-fold domains and a C-terminal steroidogenic acute regulatory protein-related lipid transfer domain (StarD14). In this study, we demonstrate that the N-terminal region of human BFIT2 (hBFIT2) constitutes a mitochondrial location signal sequence, which undergoes mitochondrion-dependent posttranslational cleavage. The mature hBFIT2 is shown to be located in the mitochondrial matrix, whereas the paralog "cytoplasmic acetyl-CoA hydrolase" (CACH, also known as ACOT12) was found in the cytoplasm. In vitro activity analysis of full-length hBFIT2 isolated from stably transfected HEK293 cells demonstrates selective thioesterase activity directed toward long chain fatty acyl-CoA thioesters, thus distinguishing the catalytic function of BFIT2 from that of CACH. The results from a protein-lipid overlay test indicate that the hBFIT2 StarD14 domain binds phosphatidylinositol 4-phosphate.
Developmental Neuroscience, 2009
Early developmental treatment of rats with 3,4-methylenedioxymethamphetamine (MDMA) was previousl... more Early developmental treatment of rats with 3,4-methylenedioxymethamphetamine (MDMA) was previously found to cause an abnormal pattern of forebrain serotonergic axon density in adulthood consisting of a cortical hypoinnervation and a striatal hyperinnervation. The present study tested the hypothesis that this reorganization was due to regional differences in brain-derived neurotrophic factor (BDNF) expression. Rats received MDMA (10 mg/kg, s.c., b.i.d.) on postnatal days (PD) 1-4, after which brain tissues were collected on PD 11, 30, and 67 for analysis. BDNF protein levels were found to be elevated in the occipital cortex but not in the hippocampus or striatum following MDMA administration. Serotonin transporter binding (an index of serotonergic fiber integrity) was significantly reduced in the hippocampus at PD 11 but returned to normal by PD 30, whereas the cortex exhibited a delayed reduction that was not manifested until PD 30. These results do not support the view that a region-specific enhancement in BDNF expression mediates the abnormal serotonergic reinnervation observed following neonatal MDMA exposure.
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Papers by Jeremiah Farelli