Chi H. Trinh

Crystal structure of monomeric human β-2-microglobulin reveals clues to its amyloidogenic properties

Dissociation of human β-2-microglobulin (β2m) from the heavy chain of the class I HLA complex is a critical first step in the formation of amyloid fibrils from this protein. As a consequence of renal failure, the concentration of circulating monomeric β2m increases, ultimately leading to deposition of the protein into amyloid fibrils and development of the disorder, dialysis-related amyloidosis. Here we present the crystal structure of a monomeric form of human β2m determined at 1.8-Å resolution that reveals remarkable structural changes relative to the HLA-bound protein. These involve the restructuring of a β bulge that separates two short β strands to form a new six-residue β strand at one edge of this β sandwich protein. These structural changes remove key features proposed to have evolved to protect β sheet proteins from aggregation [Richardson, J. & Richardson, D. (2002) Proc. Natl. Acad. Sci. USA 99, 2754–2759] and replaces them with an aggregation-competent surface. In combination with solution studies using 1H NMR, we show that the crystal structure presented here represents a rare species in solution that could provide important clues about the mechanism of amyloid formation from the normally highly soluble native protein.

Antibody fragment Fv4155 bound to two closely related steroid hormones: the structural basis of fine specificity.

BACKGROUND The concentration of steroid glucuronides in serial samples of early morning urine (EMU) can be used to predict the fertile period in the female menstrual cycle. The monoclonal antibody 4155 has been used as a convenient means of measuring the concentration of steroid glucuronides in EMU, as it specifically recognises the steroid hormone estrone beta-D-glucuronide (E3G), with very high affinity, and the closely related hormone estriol 3-(beta-d-glucuronide) (EI3G), with reduced affinity. Although 4115 binds these hormones with different affinities, EI3G differs from E3G only in the addition of a hydroxyl group and reduction of an adjacent carbonyl. To investigate the structural basis of this fine binding specificity, we have determined the crystal structures of the variable fragment (Fv) of 4155 in complex with each of these hormones. RESULTS Two crystal forms of the Fv4155-EI3G complex, at resolutions of 2.1 A and 2.5 A, and one form of the Fv4155-E3G complex, at 2.1 A resolution were solved and refined. The crystal structures show the E3G or EI3G antigen lying in an extended cleft, running form the centre of the antibody combining site down one side of the variable domain interface, and formed almost entirely from residues in the heavy chain. The binding cleft lies primarily between the heavy chain complementarity determining regions (CDRs), rather than in the interface between the heavy and light chains. In both complexes the binding of the glucuronic sugar, and rings A and B of the steroid, is specified by the shape of the narrow cleft. Analysis of the Fv structure reveals that five of the six CDR regions can be assigned to one of the predefined canonical structural classes. CONCLUSIONS The difference in the binding affinity of Fv4155 for the two steroid hormones is accounted for by a subtle combination of a less favoured hydrogen-bond geometry, and a minor rearrangement of the water molecule network around the binding site. The rearrangement of water molecules results from the burial of the additional hydroxyl group of the EI3G in a hydrophobic environment.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation.

Significance Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children; however, no vaccine exists and current immunoprophylaxis regimes are both expensive and incompletely protective. We report the crystal structure of the HRSV M2-1 transcription factor that is essential for virus gene expression and thus growth. This structure reveals how M2-1 forms an extremely stable tetramer and has allowed us to pinpoint the location of critical regions that regulate M2-1 activity, providing insight into its function. This structure may represent a potent target for new antiviral compounds. The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

Genetic Analysis of Physcomitrella patens Identifies ABSCISIC ACID NON-RESPONSIVE, a Regulator of ABA Responses Unique to Basal Land Plants and Required for Desiccation Tolerance

ABA-mediated desiccation tolerance in P. patens depends on a trimodular protein kinase, found only in early land plants and aquatic algae, proposed to have facilitated plant colonization of land. The anatomically simple plants that first colonized land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA nonresponsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain, and a C-terminal MAPKKK-like domain. anr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA, or osmotic stress and do not acquire ABA-dependent desiccation tolerance. The crystal structure of the PAS domain, determined to 1.7-Å resolution, shows a conserved PAS-fold that dimerizes through a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA nonresponsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA-responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae but are absent from more recently diverged vascular plants. We propose that ANR genes represent an ancestral adaptation that enabled drought stress survival of the first terrestrial colonizers but were lost during land plant evolution.

Exploring the roles of the metal ions in Escherichia coli copper amine oxidase.

To investigate the role of the active site copper in Escherichia coli copper amine oxidase (ECAO), we initiated a metal-substitution study. Copper reconstitution of ECAO (Cu-ECAO) restored only ∼12% wild-type activity as measured by kcat(amine). Treatment with EDTA, to remove exogenous divalent metals, increased Cu-ECAO activity but reduced the activity of wild-type ECAO. Subsequent addition of calcium restored wild-type ECAO and further enhanced Cu-ECAO activities. Cobalt-reconstituted ECAO (Co-ECAO) showed lower but significant activity. These initial results are consistent with a direct electron transfer from TPQ to oxygen stabilized by the metal. If a Cu(I)-TPQ semiquinone mechanism operates, then an alternative outer-sphere electron transfer must also exist to account for the catalytic activity of Co-ECAO. The positive effect of calcium on ECAO activity led us to investigate the peripheral calcium binding sites of ECAO. Crystallographic analysis of wild-type ECAO structures, determined in the presence and absence of EDTA, confirmed that calcium is the normal ligand of these peripheral sites. The more solvent exposed calcium can be easily displaced by mono- and divalent cations with no effect on activity, whereas removal of the more buried calcium ion with EDTA resulted in a 60−90% reduction in ECAO activity and the presence of a lag phase, which could be overcome under oxygen saturation or by reoccupying the buried site with various divalent cations. Our studies indicate that binding of metal ions in the peripheral sites, while not essential, is important for maximal enzymatic activity in the mature enzyme.

Reaction mechanism of N-acetylneuraminic acid lyase revealed by a combination of crystallography, QM/MM simulation, and mutagenesis.

N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C–C bond-forming pathway will allow further development of these enzymes for stereospecific synthesis of new enzyme products.

Molecular analysis of sixteen unrelated factor XIIIA deficient families from south‐east of Iran

high-risk myeloma. A major goal of our study was to demonstrate reproducible methods to establish various, clinically relevant myeloma cell lines (Li et al, 2007). We have recently described that both hyperdiploid and nonhyperdiploid cases were equally common (Zhan et al, 2006). We also believe that the two cases from which the LD and CF lines were established, derived from advanced hyperdiploid myelomas that turned to a proliferation signature over time. CF cells but not LD cells had partial trisomies associated with hyperdiploid myeloma. Although karyotype analysis did not show the classic trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21, the gene expression profile clearly indicated that many genes overexpressed in these two cases were from those chromosomes (data not shown). This may be due to the sensitive molecular gene expression profiling used to detect the genetic abnormalities. From 351 TT2 dataset, 6% of the myelomas in both hyperdipoid (HY) and proliferation (PR) groups were found in the group showing the lowest 10% TP53 expression (F. Zhan and J. Shaughnessy, unpublished observations), indicating that hyperdiploid myeloma could also harbor 17p13 deletion with low TP53 expression. Furthermore, DKK1, which is typically associated with hyperdiploid myeloma, was highly expressed in both LD and CF cell lines. Although recent studies have identified subtypes of HY myelomas with poor prognosis (Zhan et al, 2006; Chng et al, 2007), high expression of certain genes by LD and CF cells (e.g. CXCR4) and their growth characteristics ex vivo and in our animal models indicate that these cells are highly dependent on the bone marrow microenvironment, a typical feature of hyperdiploid myeloma. We believe that the procedures used in our work will result in the establishment of additional useful hyperdiploid cell lines.

Structures of alternatively spliced isoforms of human ketohexokinase.

The structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and peripheral KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP have been solved. The differences between KHK-A and KHK-C resulting from the spliced region are subtle and affect thermostability and probably flexibility; the mutations causing fructosuria were modelled.

Structural Insights into the Recovery of Aldolase Activity in N‐Acetylneuraminic Acid Lyase by Replacement of the Catalytically Active Lysine with γ‐Thialysine by Using a Chemical Mutagenesis Strategy

Chemical modification has been used to introduce the unnatural amino acid γ‐thialysine in place of the catalytically important Lys165 in the enzyme N‐acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site‐directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ‐thialysine through dehydroalanine (Dha) as demonstrated by ESI‐MS. Initial kinetic characterisation showed that the protein containing γ‐thialysine regained 17 % of the wild‐type activity. To understand the reason for this lower activity, we solved X‐ray crystal structures of the wild‐type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165‐γ‐thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ‐thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH‐activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine‐containing enzyme showed almost 30 % of the activity of the wild‐type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid‐containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pKa of the residue at position 165 is perturbed by replacement with γ‐thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL.

Purification, crystallization and X-ray structures of the two manganese superoxide dismutases from Caenorhabditis elegans.

Two manganese superoxide dismutase enzymes isolated from the eukaryote C. elegans have been characterized and their structures determined. The closely related structures reveal a striking similarity to manganese superoxide dismutase found in humans.