John F. Domsic

Entrapment of carbon dioxide in the active site of carbonic anhydrase II

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.

Neutron Structure of Human Carbonic Anhydrase II: Implications for Proton Transfer

Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. Despite many high-resolution X-ray crystal structures, mutagenesis, and kinetic data, the structural details of the active site, especially the proton transfer pathway, are unclear. A large HCA II crystal was prepared at pH 9.0 and subjected to vapor H-D exchange to replace labile hydrogens with deuteriums. Neutron diffraction studies were conducted at the Protein Crystallography Station at Los Alamos National Laboratory. The structure to 2.0 A resolution reveals several interesting active site features: (1) the Zn-bound solvent appearing to be predominantly a D(2)O molecule, (2) the orientation and hydrogen bonding pattern of solvent molecules in the active site cavity, (3) the side chain of His64 being unprotonated (neutral) and predominantly in an inward conformation pointing toward the zinc, and (4) the phenolic side chain of Tyr7 appearing to be unprotonated. The implications of these details are discussed, and a proposed mechanism for proton transfer is presented.

Structure and dynamics of Adeno-Associated Virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking

ABSTRACT The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus.

Genomic evolution in a virus under specific selection for host recognition

Genetic variation in viral structural proteins is often explained by evolutionary escape of strong host defenses through processes such as immune evasion, host switching, and tissue tropism. An understanding of the mechanisms driving evolutionary change in virus surface proteins is key to designing effective intervention strategies to disease emergence. This study investigated the predictability of virus genomic evolution in response to highly specific differences in host receptor structure. The bacteriophage PhiX174 was evolved on three E. coli mutant hosts, each differing only by a single sugar group in the lipopolysaccharides, used for phage attachment. Large phage populations were used in order to maximize the amount of sequence space explored by mutation, and thus the potential for parallel evolution. Repeatability was assessed by genome sequencing of multiple isolates from endpoint populations and by fitness of the endpoint population relative to its ancestor. Evolutionary lines showed similar magnitudes of fitness increase between treatments. Only one mutation, occurring in the internal DNA pilot protein H, was completely repeatable, and it appeared to be a necessary stepping stone toward further adaptive change. Substitutions in the surface accessible major capsid protein F appeared to be involved in capsid stability rather than specific interactions with host receptors, suggesting that non-specific alterations to capsid structure could be an important component of adaptation to novel hosts. 33% of mutations were synonymous and showed evidence of selection on codon usage. Lastly, results supported previous findings that evolving populations of small ssDNA viruses may maintain relatively high levels of genetic variation.

High-resolution structure of human carbonic anhydrase II complexed with acetazolamide reveals insights into inhibitor drug design.

The crystal structure of human carbonic anhydrase II (CA II) complexed with the inhibitor acetazolamide (AZM) has been determined at 1.1 A resolution and refined to an R(cryst) of 11.2% and an R(free) of 14.7%. As observed in previous CA II-inhibitor complexes, AZM binds directly to the zinc and makes several key interactions with active-site residues. The high-resolution data also showed a glycerol molecule adjacent to the AZM in the active site and two additional AZMs that are adventitiously bound on the surface of the enzyme. The co-binding of AZM and glycerol in the active site demonstrate that given an appropriate ring orientation and substituents, an isozyme-specific CA inhibitor may be developed.

Sequestration of carbon dioxide by the hydrophobic pocket of the carbonic anhydrases.

The interaction between carbon dioxide (CO(2)) and the alpha-class carbonic anhydrase, human CA 2 (HCA2) exists for only a short period due to the rapid catalytic turnover by this enzyme. The fleeting nature of this interaction has led to difficulties in its direct analysis, with previous studies placing the CO(2) in the hydrophobic pocket of HCA2s active site. A more precise location was determined via the crystal structure of CO(2) trapped in both wild-type (holo) and zinc-free (apo) HCA2. This provided a detailed description of the means by which CO(2) is held and orientated for optimal catalysis. This information can be extended to the beta and gamma class enzymes to help elucidate the binding mode of CO(2) in these enzymes.

Structural and Kinetic Study of the Extended Active Site for Proton Transfer in Human Carbonic Anhydrase II

The catalysis of CO(2) hydration by human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer from a zinc-bound water molecule to the proton shuttle His64. This proton transfer occurs along a hydrogen-bonded water network, leading to the proton shuttle residue His64, which in turn transfers the proton to bulk solvent. The side chain of His64 occupies two conformations in wild-type HCA II, pointing inward toward the zinc or outward toward bulk solvent. Previously, several studies have examined the roles of residues of the active site cavity that interact with the solvent-mediated hydrogen-bonded network between His64 and the zinc-bound water. Here these studies are extended to examine the effects on proton transfer by mutation at Lys170 (to Ala, Asp, Glu, and His), a residue located near the side chain of His64 but over 15 A away from the active site zinc. In all four variants, His64 is observed in the inward conformation associated with a decrease in the pK(a) of His64 by as much as 1.0 unit and an increase in the rate constant for proton transfer to as much as 4 micros(-1), approximately 5-fold larger than wild-type HCA II. The results show a significant extension of the effective active site of HCA II from the zinc-bound water at the base of the conical cavity in the enzyme to Lys170 near the rim of the cavity. These data emphasize that the active site of HCA II is extended to include residues that, at first glance, appear to be too far from the zinc to exert any catalytic effects.

Viral Reprogramming of the Daxx Histone H3.3 Chaperone during Early Epstein-Barr Virus Infection

ABSTRACT Host chromatin assembly can function as a barrier to viral infection. Epstein-Barr virus (EBV) establishes latent infection as chromatin-assembled episomes in which all but a few viral genes are transcriptionally silent. The factors that control chromatin assembly and guide transcription regulation during the establishment of latency are not well understood. Here, we demonstrate that the EBV tegument protein BNRF1 binds the histone H3.3 chaperone Daxx to modulate histone mobility and chromatin assembly on the EBV genome during the early stages of primary infection. We demonstrate that BNRF1 substitutes for the repressive cochaperone ATRX to form a ternary complex of BNRF1-Daxx-H3.3-H4, using coimmunoprecipitation and size-exclusion chromatography with highly purified components. FRAP (fluorescence recovery after photobleaching) assays were used to demonstrate that BNRF1 promotes global mobilization of cellular histone H3.3. Mutation of putative nucleotide binding motifs on BNRF1 attenuates the displacement of ATRX from Daxx. We also show by immunofluorescence combined with fluorescence in situ hybridization that BNRF1 is important for the dissociation of ATRX and Daxx from nuclear bodies during de novo infection of primary B lymphocytes. Virion-delivered BNRF1 suppresses Daxx-ATRX-mediated H3.3 loading on viral chromatin as measured by chromatin immunoprecipitation assays and enhances viral gene expression during early infection. We propose that EBV tegument protein BNRF1 replaces ATRX to reprogram Daxx-mediated H3.3 loading, in turn generating chromatin suitable for latent gene expression. IMPORTANCE Epstein-Barr Virus (EBV) is a human herpesvirus that efficiently establishes latent infection in primary B lymphocytes. Cellular chromatin assembly plays an important role in regulating the establishment of EBV latency. We show that the EBV tegument protein BNRF1 functions to regulate chromatin assembly on the viral genome during early infection. BNRF1 alters the host cellular chromatin assembly to prevent antiviral repressive chromatin and establish chromatin structure permissive for viral gene expression and the establishment of latent infection.

Structure determination of the cancer-associated Mycoplasma hyorhinis protein Mh-p37.

The crystal structure of the Mycoplasma hyorhinis protein Mh-p37 has been solved and refined to 1.9 A resolution. This is the first de novo structure to be determined using the recently described heavy-atom reagent [Beck et al. (2008), Acta Cryst. D64, 1179-1182] 5-amino-2,4,6-triiodoisophthalic acid (I3C), which contains three I atoms arranged in an equilateral triangle, by SIRAS methods. Data collection was performed in-house at room temperature. SHELXD and SHELXE were used to determine the I-atom positions and phase the native protein and PHENIX AutoBuild software was used to automatically fit the amino-acid sequence to the electron-density map. The structure was refined using SHELX97 to an R(cryst) of 18.6% and an R(free) of 24.0%. Mh-p37 is an alpha/beta protein with two well defined domains which are separated by a deep cleft. An unanticipated ligand bound in the center of the molecule at the base of the cleft has been modeled as thiamine pyrophosphate or vitamin B(1). Retrospective attempts to solve the crystal structure by Patterson search methods using either isomorphous or anomalous differences failed. Additionally, attempts to use proteins with the highest structural homology in the Protein Data Bank to phase the data by molecular replacement were unsuccessful, most likely in hindsight because of their poor structural agreement. Therefore, the I3C reagent offers an alternative, quick and inexpensive method for in-house phasing of de novo structures where other methods may not be successful.

Menin and Daxx Interact to Suppress Neuroendocrine Tumors through Epigenetic Control of the Membrane Metallo-Endopeptidase

Neuroendocrine tumors (NET) often harbor loss-of-function mutations in the MEN1 and DAXX tumor suppressor genes. Here, we report that the products of these genes, menin and Daxx, interact directly with each other to suppress the proliferation of NET cells, to a large degree by inhibiting expression of the membrane metallo-endopeptidase (MME). Menin and Daxx were required to enhance histone H3 lysine9 trimethylation (H3K9me3) at the MME promoter, as mediated partly by the histone H3 methyltransferase SUV39H1. Notably, the menin T429K mutation associated with a NET syndrome reduced Daxx binding, MME repression, and proliferation of NET cells. Conversely, inhibition of MME in NET cells repressed proliferation and tumor growth in vivo Our findings reveal a previously unappreciated cross-talk between two crucial tumor suppressor genes thought to work by independent pathways, focusing on MME as a common target of menin/Daxx to treat NET. Cancer Res; 77(2); 401-11. ©2016 AACR.