A posteriori design of crystal contacts to improve the X-ray diffraction properties of a small RNA enzyme

Insertion of a dangling 5′-uracil and incorporation of synthetic linkers at the domain interface of a minimal hairpin ribozyme have been investigated as means of favorably influencing crystal packing. These modifications lead to changes in the ribozyme’s structural elements that mimic packing within a natural four-way helical junction, thereby providing an example of how knowledge-based design can be used to enhance the diffraction properties of a tertiarily folded RNA

Three-dimensional structure of galactose-1-phosphate uridyltransferase from Escherichia coli at 1.8 Å resolution

ABSTRACT: Galactose-1-phosphate uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDPgalactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multi le isomorphous replacement and electron density modification techniques and has "half-barrel". The barrel staves are formed by nine strands of antiparallel P-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the halfbarrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, Sy of a cysteine is modified by P-mercaptoethanol, which prevents the a-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Led4, Phe75, Am7', Asp78, Phe79, and Va11°8. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations. been refined to 1.8 x resolution. Enzyme subunits consist of a single domain with the topology of a Galactose-1 -phosphate uridylyltransferase (hexose-1 -phosphate uridylyltransferase, EC 2.7.7.12) catalyzes the nucleotide exchange between UDP-hexoses and hexose 1-phosphates. This provides an essential balance of UDPglc' and UDP-gal for the cell. Such activated hexose sugars are consumed in the synthesis of disaccharides, glycoproteins, glycolipids, and glycoge

Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure

Riboswitches are structural elements in the 50 untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ1 (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ1 riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at nearphysiological Mg2+ concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Go¯ -model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers0 pre-folded states and their ligand binding affinities

Structural Studies of Transition-State Stabilization by a Small Catalytic RNA, Metabolite-Binding by a Regulatory RNA Sequence and the Mechanism of Action of Activation Induced Deaminase.

Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.Structural knowledge of an enzyme’s architecture can help elucidate the details of how it performs its biological role at the molecular level. In a case study of the hairpin ribozyme, structures were solved to high resolution in complex with reaction-intermediate analogs revealing key chemical groups in catalytically-relevant configurations. Importantly, the non-bridging oxygens of the scissile phosphate were tightly restrained in a conformation apparently promoting an inductive effect to elevate the pKa of a crucial active-site nucleobase while also ameliorating the unfavorable buildup of negative charge at the leaving group during the phosphoryl-transfer reaction. Active-site water molecules were ascribed a new role in both geometric and electrostatic stabilization of the reaction intermediate. By comparison to proteinaceous ribonuclease structures harboring the same reaction-intermediate analog, the catalytic strategies employed by these two evolutionarily-disparate enzyme classes appear to be a case of convergent evolution. In a second investigation, the aptamer domain of a metabolite-sensing non-coding regulatory RNA sequence was analyzed. Construct design, initial crystallization and attempts to solve the structure are described followed by improved methods to prepare a homogeneous RNA-metabolite complex. The resulting complex was evaluated by dynamic light scattering as well as small angle X-ray scattering. Subsequent optimization of crystallization conditions and diffraction analyses are also reported. The final area of focus encompasses biochemical and computational efforts to probe the mechanism of action of activation induced deaminase (AID). AID is a member of the cytidine deaminase superfamily and functions in the immunoglobulin maturation processes of somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Despite considerable research, questions remain regarding how AID is targeted to its substrate(s), what other cellular cofactors are necessary for function and how its activity is regulated since dysfunction or misregulation of AID has the potential to cause cancer. Efforts to crystallize AID were complemented by computational modeling, which has provided the basis for targeted biochemical studies aimed at answering outstanding questions. Perspectives on the success and pitfalls of continued modeling are discussed in light of recent empirical structural information reported for related family members

Thermodynamic Analysis and Characterization of Viral-Host Protein Interactions as a Preface to HIV Therapeutic Design

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2013.The infection and propagation of the human immunodeficiency virus type 1 (HIV- 1) within a host depends on successful interactions between viral and cellular proteins. In some cases the virus has evolved pathways to hijack the cellular machinery in order to evade the innate immune factors that would otherwise restrict viral infectivity. For example, the HIV-1 auxiliary protein Vif plays an essential role in host-cell infection by suppressing the activity of the host’s innate antiretroviral proteins in the APOBEC3 (A3) family. The mechanism of action for Vif entails binding to A3 proteins in order to recruit them to the host’s own Cullin-RING E3 ubiquitin ligase (CRL), resulting in their ubiquitination and degradation. The CRL comprises the cellular proteins Cullin5 (Cul5), ElonginB, ElonginC (EloB/C), and Rbx2. Recently the human protein CBFβ was found to be essential for HIV-1 infectivity and it is indispensible for Vif recruitment of A3 proteins to the CRL. At present, the role of CBFβ within the CRL remains elusive. To better define the binding affinity and interfaces of various proteins present in Vif-hijacked CRL complexes I developed methods for bacterial expression and purification of: (i) CRL-like complexes comprising HIV-1 Vif/CBFβ/EloB/C, (ii) human Cul5, (iii) the Vif/CBFβ/EloB/C/Cul5 complex, and (iv) human A3C. Using isothermal titration calorimetry (ITC) I found that CBF increases the relative affinity of Cul5 binding to CRL-like complexes by 60-fold compared to CRL-like complexes in which N-terminally truncated Vif(95-192) was utilized to eliminate CBF binding. Heat capacity measurements were negative (ΔCP = -0.52) and correlated the binding of Cul5 to CRL-like complexes to a large burial of hydrophobic residues. In order to identify specific regions of HIV-1 Vif that interact with Cul5 in CRL-like complexes, the following samples were subjected to hydrogen/deuterium exchange mass spectrometry: Vif/CBFβ/EloB/C, Vif/CBFβ/EloB/C/Cul5, and Cul5. The results revealed a significant number of regions within Vif that become protected upon Cul5 binding including novel regions that have not been described in the literature. Collectively my work provides a global map for protein interactions within the pentamer complex that provides a basis for rational experimental analysis of viral-host protein interactions

Biophysical characterization of the HIV-1 viral infectivity factor and the intrinsic-immunity factor APOBEC3G

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biochemistry and Biophysics, 2010.A delicate power struggle exists between antiretroviral factors of the intrinsic immune system and the tactics evolved by retroviruses to subvert the action of these factors. In humans, one such antiretroviral factor is APOBEC3G (A3G), a cytidine deaminase capable of mutating retroviral HIV-1 DNA. This deleterious process is prevented by an HIV-1 accessory protein known as the viral infectivity factor (vif), which serves as a substrate receptor for an ElonginB­ElonginC-CullinS-SOCS-box-Rbx2 E3 ubiquitin ligase complex (ECS). The ECS targets A3G for proteasomal degradation, which makes the cognate interactions between vif and cellular proteins of great interest. In the first section of this dissertation I present a novel method for expression of an ElonginB-ElonginC-vif complex. Isothermal titration calorimetry was used to examine the thermodynamic parameters of the interaction between the preformed tripartite complex with CullinS (CulS). The results suggest that a zinc-binding motif in vif mediates CulS binding and that this interaction is different fundamentally when compared to the cellular SOCS-box proteins. ITC studies with vif mutants suggest the host-virus binding interface is malleable and capable of withstanding subtle changes in amino acid content. Analytical ultracentrifugation (AUC) was used to identify vif as a self-associating protein and to ascribe the zinc-binding domain as a primary determinant of self-association. In a second investigation, the hydrodynamic properties of A3G were modeled from AUC analysis. The results indicate that A3G self-associates, and that the enzyme in solution comprises a monomer-dimer-tetramer equilibrium. Hydrodynamic modeling and shape analyses suggested that the predominant form of A3G in solution is an elongated dimer. These characteristics have implications for A3G function and modulation of deaminase activity. Structural models of proteins serve as guides to understanding function and as blueprints for the design of therapeutics. X-ray crystallography represents the pinnacle of current structural techniques and provides models of the highest quality. As such, solving the structure of vif and CulS in complex, or in isolation, will shed light on the determinants of binding. To this end, I have crystallized the N-terminal domain of CulS, as well as a complex comprising a portion of vif bound to ElonginB and ElonginC. Diffracting crystals of CulS have been grown from 3 conditions that provide a starting point for optimization. Efforts to determine the structure of CulS crystals are discussed as well. Similarly, crystals of the ElonginB-ElonginC-vif complex have been prepared in several growth habits. The methodology used to achieve crystallization from constructs of varied length will be discussed, as well as the use of dynamic light scattering and AUC, which helped to direct my efforts

Chemical and structural analysis of RNA-based gene regulation

Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2009.Non-protein coding RNA (ncRNA), once thought to be a functional spectator in biological systems have recently been recharacterized as a critical component of many essential life processes. Due to the emergent pervasiveness of these vital biomolecules, structure/function studies aimed at elucidating their molecular basis of action are becoming increasingly important to the scientific community. To that end, this thesis is aimed at understanding the chemical and structural basis of function in ncRNA macromolecules, and to compare their molecular basis of action with proteins. In order to accomplish this, the thesis focuses on two ncRNA macromolecules: the hairpin ribozyme and the PreQ1 riboswitch. The hairpin ribozyme is an autocatalytic RNA motif found in tobacco ringspot virus satellite RNA. It serves to process concatentated RNA transcripts during rolling-circle RNA. Unlike other catalytic RNA motifs, the hairpin ribozyme relies solely on the localized nucleobases of the catalytic core. This all-RNA based mode of catalysis makes it an ideal system to elucidate the chemical principles employed by RNA for rate acceleration. To shed light on the mode of activity of this RNA construct, a synergistic approach using organic synthesis, enzymology and X-ray crystallography has been undertaken. From these studies, we have begun to understand how this RNA uses substrate orientation and functional group complimentarity to initiate activity. Additionally, parallels between RNA and protein enzymes have been derived, highlighting similar mechanisms of RNA processing. The second goal of the thesis is aimed at understanding the mode of metabolite recognition in the PreQ1 riboswitch aptamer domain. The PreQ1 riboswitch aptamer domain functions to regulate bacterial genes encoding proteins that are critical for exogenous preQ1 uptake or de novo biosynthesis. Notably, the essential aptamer domain was characterized to be ~3-times smaller than other analogous purine-sensing riboswitches. To understand how PreQ1 is recognized by the aptamer, an examination was performed employing X-Ray crystallographic techniques; this study provided a molecular glimpse into the mode of metabolite recognition. From these studies, we have expanded the knowledge of structure-based functional RNA, and have been able to compare modes of metabolite recognition between RNA and protein macromolecules

A comparison of vanadate to a 2′–5′ linkage at the active site of a small ribozyme suggests a role for water in transition-state stabilization

The potential for water to participate in RNA catalyzed reactions has been the topic of several recent studies. Here, we report crystals of a minimal, hinged hairpin ribozyme in complex with the transition-state analog vanadate at 2.05 Å resolution. Waters are present in the active site and are discussed in light of existing views of catalytic strategies employed by the hairpin ribozyme. A second structure harboring a 2′,5′-phosphodiester linkage at the site of cleavage was also solved at 2.35 Å resolution and corroborates the assignment of active site waters in the structure containing vanadate. A comparison of the two structures reveals that the 2′,5′ structure adopts a conformation that resembles the reaction intermediate in terms of (1) the positioning of its nonbridging oxygens and (2) the covalent attachment of the 2′-O nucleophile with the scissile G+1 phosphorus. The 2′,5′-linked structure was then overlaid with scissile bonds of other small ribozymes including the glmS metabolite-sensing riboswitch and the hammerhead ribozyme, and suggests the potential of the 2′,5′ linkage to elicit a reaction-intermediate conformation without the need to form metalloenzyme complexes. The hairpin ribozyme structures presented here also suggest how water molecules bound at each of the nonbridging oxygens of G+1 may electrostatically stabilize the transition state in a manner that supplements nucleobase functional groups. Such coordination has not been reported for small ribozymes, but is consistent with the structures of protein enzymes. Overall, this work establishes significant parallels between the RNA and protein enzyme worlds