Browsing by Subject "X-ray crystallography"

Gram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids while the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine-based saccharolipid that is further modified with core and O-antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. The enzymes of Lipid A biosynthesis are conserved in Gram-negative bacteria; in most species, a single copy of each bio-synthetic gene is present. The exception is lpxH, which is an essential gene encoding a membrane-associated UDP-2,3-diacylglucosamine hydrolase, which catalyzed the attack of water upon the alpha-phosphate of its substrate and the leaving of UMP, resulting in the formation of lipid X. Many Gram-negatives lack an lpxH orthologue, yet these species must possess an activity analogous to that of LpxH. We used bioinformatics approaches to identify a candidate gene, designated lpxI, encoding this activity in the model organism Caulobacter crescentus. We then demonstrated that lpxI can rescue Escherichia coli deficient in lpxH. Moreover, we have shown that LpxI possesses robust and specific UDP-2,3-diacylglucosamine hydrolase activity in vitro. We have developed high-yield purification schema for Caulobacter crescentus LpxI (CcLpxI) heterologously expressed in E. coli. We crystallized CcLpxI and determined its 2.6 Å x-ray crystal structure in complex with lipid X. CcLpxI, which has no known homologues, consists of two novel domains connected by a linker. Moreover, we have identified a point mutant of CcLpxI which co-purifies with its substrate in a 0.85:1 molar ratio. We have solved the x-ray crystal structure of this mutant to 3.0 Å; preliminary comparison with the product-complexed model reveals striking differences. The findings described herein set the stage for further mechanistic and structural characterization of this novel enzyme.

In this work, we also isolate and characterize LpxB, an essential lipid A biosynthetic gene which is conserved among all Gram-negative bacteria. We purify E. coli and Hemophilus influeznea LpxB to near-homogeneity on a 10 mg scale, and we determine that E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micellar assay system, suggesting that catalysis occurs at the lipid interface. E. coli LpxB partitions with membranes, but this interaction is partially abolished in high-salt conditions, suggesting that a significant component of LpxB's membrane association is ionic in nature. E. coli LpxB (Mr ~ 43 kDa) is a peripheral membrane protein, and we demonstrate that it co-purifies with phospholipids. We estimate, by autoradiography and mass-spectrometry, molar ratios of phospholipids to purified enzyme of 1.6-3.5:1. Transmission electron microscopy reveals the accumulation of intra-cellular membranes when LpxB is massively over-expressed. Alanine-scanning mutagenesis of selected conserved LpxB residues identified two, D89A and R201A, for which no residual catalytic activity is detected. Our data support the hypothesis that LpxB performs catalysis at the cytoplasmic surface of the inner membrane, and provide a rational starting-point for structural studies. This work contributes to knowledge of the small but growing set of structurally and mechanistically characterized enzymes which perform chemistry upon lipids.

The structural plasticity conferred by conformational flexibility has increasingly been recognized as a likely determinant of function. For example, multiscale heterogeneity in the calmodulin central helix most likely helps it in binding over 100 protein targets, and a concerted motion seen in both nuclear magnetic resonance (NMR) and crystal structures of ubiquitin is proposed to underlie its functional plasticity of promiscuous binding to many different proteins with high affinity. However, flexibility is manifested in a variety of ways, depending both on the protein itself and on how it is observed. Conformational heterogeneity (the term we use for flexibility when studied by X-ray crystallography) is evident in electron density, either as fully separated peaks or as anisotropic density shapes showing fluctuation of atom groupings. Many phenomena contribute to conformational heterogeneity in crystal structures, from diverse crystal contacts to functionally relevant conformational fluctuations on a wide range of time and size scales.

In addition to ubiquitin and calmodulin, the Staphylococcus aureus virulence factor staphylococcal protein A (SpA) is an example of a highly heterogeneous protein. SpA is a major contributor to bacterial evasion of the host immune system, through high-affinity binding to host proteins such as antibodies, von Willebrand factor, and tumor necrosis factor receptor 1 (TNFR1). The protein includes five small three-helix-bundle domains (E-D-A-B-C) separated by conserved flexible linkers. Prior attempts to crystallize individual domains in the absence of a binding partner were apparently unsuccessful. There are also no previous structures of tandem domains. In this thesis, I report the high-resolution crystal structures of a single C domain (collected at both cryogenic and room temperatures), a single A domain, and two B domains connected by the conserved linker. All four apo structures exhibit extensive multiscale conformational heterogeneity, which required novel modeling protocols. Comparison of domain structures shows that helix1 orientation is especially heterogeneous, coordinated with changes in sidechain conformational networks and contacting protein interfaces.

The interaction between a SpA domain and the Fc fragment of IgG was partially elucidated previously in the crystal structure 1FC2. Although informative, the previous structure wasn't properly folded and left many substantial questions unanswered, such as a detailed description of the tertiary structure of SpA domains in complex with Fc and the structural changes that take place upon binding. In this thesis, I report the 2.3-A structure of a fully folded SpA domain in complex with Fc. My structure indicates that there are extensive structural rearrangements necessary for binding Fc, including concerted rotamer changes and coupled backbone rearrangements that lead to a difference in helix1 angle. The conformational heterogeneity of the helix1/2 interface is also eliminated in the complex, with previously poly-rotameric interfacial residues locking into single rotamer conformations. Such a loss of conformational heterogeneity upon formation of the protein-protein interface may occur in SpA and in its multiple binding partners and may be an important structural paradigm in other functionally plastic proteins.

Medical advances often come as a result of understanding the underlying mechanisms of life. Life, in this sense, happens at various scales. A very complex and interesting one is the molecular scale. Understanding life’s mechanistic details at this level will provide the most promising therapies to modern ailments. Because of structure and function’s close relationship, knowledge of macromolecular structure provides invaluable insight into molecular mechanism.

A major tool used to get structural information at the molecular scale is X-ray crystallography. Such experiments result in an electron density map from which a model is built. Building such a model is a difficult task, especially at low resolu- tion where detailed features in the electron density deteriorate making it difficult to interpret. However, many advances in the field have greatly eased the model build- ing task, in fact, at high resolutions it has become automated. However, human inspection is still required to get a correct solution.

The largest boon to model building has been the application of structural knowl- edge. A prominent example is bond and dihedral angles. We often know what is absolutely not allowed and often convince ourselves we know everything that is al- lowed. This work focuses on the fuzzy border between allowed and disallowed. The hypothesis is that rare structural conformations exist but one needs to take great care in modeling them.

This work has two major components – rotamers (protein sidechain conformation)

and Hoogsteen base pairing in DNA. I first describe methods used to gain empirical knowledge about rotamers and how that knowledge is used in model validation. Part of this knowledge is rotamer-dependent bond angle deviations. I describe how the observation and quantitation of these deviations is used in a novel set of restraints in protein structure refinement. To provide structural context to rare rotamers, I describe where and why some occur.

My DNA work has focused on Hoogsteen base pairing. I describe a collaborative survey of existing Hoogsteen base pairs in the PDB. Lessons learned during the survey led to the other DNA topic, the detection and correction of mismodeled purines. I identified Hoogsteens in the PDB mismodeled as Watson-Crick base pairs. This work underscores that Hoogsteens are extremely rare but nonetheless do occur.

The fuzzy borderland between allowed and disallowed is a strange place filled with the most interesting structural features. My work here has focused on this area, bringing into view many rare conformations. Going forward we need to ensure that conformational frequency is taken into account during model building, refinement, and validation.

Francisella tularensis is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase pathogenicity island (FPI) that is regulated by the macrophage growth locus protein A (MglA), the stringent starvation protein A (SspA) and the pathogenicity island gene regulator (PigR). MglA•SspA•PigR interacts with the RNA polymerase (RNAP) to activate FPI transcription. The activity of MglA•SspA•PigR•RNAP is mediated by ppGpp, the alarmone of the stringent response. However, the molecular mechanisms involved in FPI transcriptional regulation are not well understood.

While most bacterial SspA proteins interact with the RNAP as a homodimer, F. tularensis SspA forms a heterodimer with MglA, which is unique to F. tularensis. To gain insight into MglA function, we performed structural and biochemical studies. The MglA structure revealed that it contains a fold similar to the SspA protein family, and unexpectedly formed a homodimer in the crystal. Chemical crosslinking and size exclusion chromatography (SEC) studies showed that, while it can self-associate in solution to form a dimer, MglA preferentially forms a heterodimer with SspA. As research with MglA highlighted that MglA•SspA is likely the only functional SspA protein in Francisella, X-ray crystallography was used to determine the structure of MglA•SspA to 2.65 Å. Analysis of the MglA•SspA structure revealed a vast hydrogen bond network at the interface that is significantly larger than the network at the MglA homodimerization interface. This difference could explain why MglA and SspA form a stable heterodimer and MglA (in the absence of SspA) forms a transient homodimer.

To gain insight into the molecular mechanism by which ppGpp mediates FPI transcription, differential radial capillary action of ligand assay (DRaCALA) was performed to determine which Francisella protein binds ppGpp. The DRaCALA experiments revealed that the MglA•SspA complex binds ppGpp specifically and with high affinity, while PigR, MglA and E. coli SspA do not. To characterize this interaction the structure of MglA•SspA was determined in complex with ppGpp (MglA•SspA•ppGpp) to 2.8 Å resolution, and revealed a single ppGpp molecule, which is also coordinated by Mg2+, bound per heterodimer. Analysis of structure-guided MglA•SspA ppGpp-binding mutants by DRaCALA confirmed the crystallographic binding site of ppGpp. Intriguingly, the binding of ppGpp does not produce a marked conformational change in MglA•SspA, but as the ppGpp-binding site overlaps with the previously ascribed PigR interaction surface it seems likely that ppGpp mediates the interaction between MglA•SspA and PigR.

The interaction between PigR and MglA•SspA is largely uncharacterized. Using fluorescence anisotropy we sought to determine which portion of PigR is responsible for interactions with MglA•SspA. Using fluoresceinated PigR peptides we determined that the final 22 residues of the PigR C-terminus interact with MglA•SspA. These results were corroborated using bridge-hybrid experiments by our collaborators. Further, as FA performed with and without ppGpp shows no difference in affinity between MglA•SspA and PigR, ppGpp must mediate this interaction through an as of yet undetermined mechanism, which is supported by very different millipolarization values indicating perhaps alternative binding modes of PigR.

All together, our results contribute significantly to our understanding of FPI transcriptional regulation.

Lipopolysaccharide, the physical barrier that protects Gram-negative bacteria from various antibiotics and environmental stressors, is anchored to the outer membrane by the phosphorylated, acylated disaccharide of glucosamine known as lipid A. Besides being necessary for the viability of most Gram-negative bacteria, lipid A interacts directly with specific mammalian immune cell receptors, causing an inflammatory response that can result in septic shock. The lipid A biosynthetic pathway contains nine enzymatic steps, the sixth being the phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) precursor to form lipid IV_A by the inner membrane-bound kinase LpxK, a divergent member of the P-loop containing nucleotide triphosphate hydrolase superfamily. LpxK is the only known P-loop kinase to act on a lipid at the membrane interface.

We report herein multiple crystal structures of Aquifex aeolicus LpxK in apo as well as ATP, ADP/Mg2+, AMP-PCP, and chloride-bound forms. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other P-loop kinase family members, is responsible for catalysis at the P-loop and positioning of the DSMP substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate using a 25º hinge motion about its base which also assembles the necessary catalytic residues at the active site.

Using a thin-layer chromatography-based radioassay, we have performed extensive kinetic characterization of the enzyme and demonstrate that LpxK activity in vitro is dependent on the presence of detergent micelles, the use of divalent cations, and formation of a ternary LpxK-ATP/Mg2+-DSMP complex. Implementing steady-state kinetic analysis of multiple point mutants, we identify crucial active site residues. We propose that the interaction of D99 with H261 acts to increase the pK_a of the imidazole group, which in turn serves as the catalytic base to deprotonate the 4’-hydroxyl of DSMP. An analogous mechanism has not yet been reported for any member of the P-loop kinase family.

The membrane/lipid binding characteristics of LpxK have also been also investigated through a crystal structure of the LpxK-lipid IV_A product complex along with point mutagenesis of residues in the DSMP binding pocket. Critical contacts with the bound lipid include interactions along the glucosamine backbone and the 1-position phosphate group, especially through R171. Furthermore, analysis of truncation mutants of the N-terminal helix of LpxK demonstrates that this substructure is a critical hydrophobic contact point with the membrane, and that both charge-charge and hydrophobic interactions contribute to the localization of LpxK at the lipid bilayer.

Overall, this work has contributed significantly to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates. It has also provided insight into the process of enzyme evolution as LpxK, while containing a similar core domain as other P-loop kinases, has developed multiple subdomains required for both cellular localization and recognition of novel substrates. Finally, the presence of multiple crystal structures and detailed understanding of the LpxK catalytic mechanism will improve the chances of successfully targeting this essential step in lipid A biosynthesis in the pursuit of novel antimicrobials.

The bacterial protein Hfq is an RNA chaperone and pleiotropic posttranscriptional regulator. Hfq binds to A and U-­‐‑rich regions of small regulatory RNA (sRNA) to their cognate mRNA to facilitate their annealing, affecting stability and translation. The protein is involved in the regulation of a wide array of cellular processes, including many related to environmental stress response and virulence. The importance of Hfq in Gram-­‐‑negative bacteria is well understood, while a less clear picture remains for Gram-­‐‑positive species. We have determined the structure of Hfq from the Gram-­‐‑positive pathogen Listeria monocytogenes (Lm) in its apo form and bound to U6 RNA. U6 RNA binds to the proximal face in a canonical manner but with additional contacts made to the N3 and O4 positions of uridine by residue Q6 of Hfq. Furthermore, fluorescence polarization and tryptophan fluorescence quenching (TFQ) reveal that U16 RNA binds to Hfq with higher affinity than U6, on the basis of the longer sequence’s ability to simultaneously bind in the proximal pore and the lateral rim of the protein. TFQ also shows that surprisingly Lm Hfq can accommodate (GU)3G and U6 RNA on both proximal and distal face binding sites, suggesting Lm Hfq has a less stringent distal face A-­‐‑site than previously reported for Hfq from other species.

To understand fully how sRNA bind to the proximal face and are positioned to anneal with mRNA, we have attempted to crystallize U16 RNA with Lm Hfq and fragments of sRNA containing a hairpin with a poly-­‐‑U tail with both Lm and Escherichia coli (Ec) Hfq. While this endeavor has been largely fruitless, we have determined the structure of Ec Hfq with dsDNA. Ec Hfq-­‐‑DNA binding has been observed in multiple studies but the molecular mechanism of recognition of this nucleic by Hfq is unknown. The DNA binds to the proximal face with conserved lateral rim residues N13, R16, and R17, and residue Q41 contacting the phosphate backbone. Fluorescence polarization and TFQ reveal both dsDNA and dsRNA bind to the proximal face, indicating the observed DNA binding mode may actually be a double stranded nucleic acid binding site. We have thusly proposed a model in which the proximal face of Hfq stabilizes both single and double stranded portions of sRNA, positioning it appropriately for formation of an Hfq-­‐‑sRNA-­‐‑mRNA ternary complex.

Toxin-­‐‑antitoxin (TA) modules are ubiquitous among bacterial species with bioinformatics studies identifying at least 10000 putative TA modules. These modules have diverse functions and are implicated in many processes, including gene regulation, stress response, and persister cell formation. Whereas many bacteria may have only a handful of TA modules, the genome of Mycobacterium tuberculosis (Mtb) contains 79 TA modules, 37 of which have been confirmed to be functional in vivo. A recent transcriptome analysis of Mtb persister cells revealed 10 up-­‐‑regulated TA modules. Four of these modules do not belong to a previously characterized TA family. We have determined the structure of a C-­‐‑terminally truncated version of the toxin Rv3189 (1-­‐‑164 of 206 amino acid residues) in complex with the anti-­‐‑toxin Rv3188. Rv3189 is structurally homologous to the ADP-­‐‑ribosyltransferase core domain, suggesting a never before observed mode of action for a TA module. The toxin has been shown to inhibit growth in an E. coli model with structure guided mutagenesis identifying residues that are critical for toxin function.

The biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates membrane transport of oligosaccharide precursors from their cytoplasmic site of synthesis to their site of assembly outside the cytoplasm. To address this problem, cells utilize transporters such as those of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily to flip lipid-linked oligosaccharides across the cytoplasmic membrane. The MOP superfamily member MurJ has been shown to be the flippase that transports the lipid-linked peptidoglycan precursor lipid II, but the lack of structural information has limited our mechanistic understanding of the MurJ transport cycle. We determined the first crystal structure of MurJ (MurJTA from Thermosipho africanus) to 2.0-Å resolution, which assumed an inward-facing conformation unlike all other outward-facing structures of MOP transporters. Our structural and mutagenesis studies provide insight into a putative model of lipid II binding and an alternating-access mechanism of transport.

The emergence of multidrug-resistant nosocomial Gram-negative (GN) pathogens has become a major public health threat. Carbapenem-resistant P. aeruginosa, A. baumannii, and extended-spectrum--lactamase (ESBL)-producing Enterobacteriaceae are the top three pathogens that pose the greatest threat to human health in the 2017 WHO report [1]. This alarming list highlights the urgent need to develop new antibiotics, preferably by targeting novel pathways in these bacteria, for which existing resistance mechanisms are lacking. GN bacteria are characterized by enrichment of lipid A-anchored LPS or LOS in the outer monolayer of their outer membrane. Lipid A biosynthesis represents a highly conserved pathway that has never been exploited by commercial antibiotics. Targeted inhibition of the first committed step of lipid A biosynthesis, LpxC, has proven to be a viable route for antibiotic development. However, current efforts have been hindered by unexpected cardiovascular toxicity in vivo. Herein, we report the preclinical characterization of a novel LpxC inhibitor that has slow, tight binding, low pM affinity, oral bioavailability, and an exceptional in vivo safety profile. Additionally, accumulating evidence from our recent studies shows that inhibition of lipid A enzymes downstream of LpxC, such as LpxH, kills bacteria not only by disrupting the essential lipid A biosynthesis, but also by accumulating toxic lipid A intermediates, thus delivering a double punch for bacteria [2, 3]. Our preliminary structural characterization of AZ1 in complex with LpxH has led to the development of more potent inhibitors. By conducting an in-depth structural analysis of LpxH in complex with several generations of AZ1-based inhibitors, we have not only gleaned vital knowledge about the electrostatic forces dictating inhibitor recognition of LpxH, but also developed a suitable candidate for in vivo trials that displays a good safety profile and has a preliminary rate of 80% in recovering mice from lethal infections of Klebsiella pneumoniae.

Cancer is one of the most devastating diseases in modern society, with over 1.6 million new cancer cases occurring in the US alone each year. DNA-damaging agents are often the first line of defense against rapidly dividing cancer cells. However, cancer cells can become resistant to chemotherapy by up-regulating an error-prone DNA-repair process called translesion DNA synthesis (TLS). The Rev1 polymerase orchestrates this pathway by recruiting one of three inserter polymerases and the extender polymerase (Pol ζ) to bypass the lesion. Here we report the discovery and characterization of an inhibitor of the protein-protein interaction between Rev1 and Rev7, a subunit of Pol ζ, using biochemical and biophysical techniques. Our X-ray crystallographic structural analysis of the Rev1 and the inhibitor (JH-RE-06) complex reveals that the inhibitor blocks Rev7 binding by inducing Rev1 dimerization. Such an unexpected observation is confirmed by an in vitro crosslinking assay. In vitro cell-killing assays show that JH-RE-06 enhances sensitivity of a variety of cancer cell lines to a wide range of chemotherapeutic agents; furthermore, co-administration of JH-RE-06 with cisplatin significantly suppresses melanoma growth in mice and prolongs the survival time of tumor bearing mice, highlighting the therapeutic potential of translesion synthesis inhibitors as a novel class of cancer adjuvant therapeutics to enhance the outcome of chemotherapy currently available to cancer patients.

Due to their compromised immune systems, cancer patients are particularly susceptible to opportunistic bacterial infections, many of which are becoming rapidly resistant to current antibiotic therapies. We describe the combined use of X-ray crystallography and NMR spectroscopy to delineate a cryptic inhibitor envelope for optimization of a small molecule inhibitor of LpxC, an enzyme essential to the survival of Gram-negative bacteria. The resulting inhibitor shows vast improvement over its parent compound over a wide range of bacterial orthologs.

In summary, we demonstrate successful structural characterization and structure-guided design and optimization of lead compounds in two different systems. These studies have profound implications for drug discovery and lead optimization in other disease-relevant systems as well.