The capsid proteins of two flaviviruses, yellow fever virus and dengue virus, were expressed in Escherichia coli and purified to near homogeneity suitable for biochemical characterization and structure determination by nuclear magnetic resonance. The oligomeric properties of the capsid protein in solution were investigated. In the absence of nucleic acid, both proteins were predominately dimeric in solution. Further analysis of both proteins with far-UV circular dichroism spectroscopy indicated that they were largely alpha-helical. The secondary structure elements of the dengue virus capsid were determined by chemical shift indexing of the sequence-specific backbone resonance assignments. The dengue virus capsid protein devoid of its C-terminal signal sequence was found to be composed of four alpha helices. The longest alpha helix, 20 residues, is located at the C terminus and has an amphipathic character. In contrast, the N terminus was found to be unstructured and could be removed without disrupting the structural integrity of the protein.The Flaviviridae family of enveloped RNA viruses causes significant disease in both humans and agriculturally important animals. Flavivirus, the largest of the three genera of Flaviviridae, comprises over 70 viruses, mostly arthropod transmitted, including yellow fever virus (YF), dengue virus (DEN), West Nile virus, and tick-borne encephalitis virus (TBE) (15). The mature flavivirus particle is spherical with a diameter of 50 nm and contains multiple copies of three different structural proteins (C, M, and E), a host-derived membrane bilayer, and a single copy of a positive-sense RNA genome of approximately 11,000 nucleotides. The RNA genome is translated from a single open reading frame generating a polyprotein that is processed by viral and host proteases to yield the three structural proteins located at the N terminus followed by at least seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (23). The nonstructural proteins associate to form the viral replicase complex, for which several enzymatic functions have been identified, including protease, helicase, methyltransferase, and RNA-dependent RNA polymerase (15). The replicase complex is associated with intracellular membranes of the infected host and induces discrete membrane structures (16). Recent evidence suggests that the process of particle assembly may be coupled to genome replication (6,12). This coupling of replication and virus assembly has made analysis of flavivirus assembly difficult, and thus, little is known about this important aspect of the virus life cycle.The structure of DEN, recently determined by cryoelectron microscopy (cryo-EM) and three-dimensional image reconstruction, has elucidated the molecular organization of the end product of the flavivirus assembly pathway (11). The organization of the E protein within the DEN particle was determined by modeling the atomic resolution structure of TBE E protein (22) into the outer density of the cryo-EM reconstruction. The model consist...

The reported structures of many CC chemokines show a conserved dimer interface along their N-terminal region, raising the possibility that the quaternary arrangement of these small immune proteins might influence their function. We have produced and analyzed several mutants of MIP-1 beta having a range of dimer K(d) values in order to determine the significance of dimerization in receptor binding and cellular activation. NMR and analytical ultracentrifugation were used to analyze the oligomeric state of the mutants. Functional relevance was determined by receptor binding affinity and the ability to invoke intracellular calcium release from CHO cells transfected with the MIP-1 beta receptor CCR5. The monomeric N-terminally truncated mutant MIP(9) was able to bind the CCR5 receptor with a K(i) of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability to tightly bind (K(i) = 480 pM) and to activate (EC(50) = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP-1 beta, as a key determinant for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic side chain to be important for both binding to CCR5 and chemokine dimerization.

The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C(+)-O- charge separation that was seen previously with the mammalian enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C = O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C V O and the protonated His-195. Thus, while the C = O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stability substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 in vitro and in vivo .

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The viral nucleocapsid (N) protein plays an essential role in viral RNA packaging. In this study, recombinant SARS-CoV N protein was shown to be dimeric by analytical ultracentrifugation, size exclusion chromatography coupled with light scattering, and chemical cross-linking. Dimeric N proteins selfassociate into tetramers and higher molecular weight oligomers at high concentrations. The dimerization domain of N was mapped through studies of the oligomeric states of several truncated mutants. Although mutants consisting of residues 1-210 and 1-284 fold as monomers, constructs consisting of residues 211-422 and 285-422 efficiently form dimers. When in excess, the truncated construct 285-422 inhibits the homodimerization of full-length N protein by forming a heterodimer with the full-length N protein. These results suggest that the N protein oligomerization involves the C-terminal residues 285-422, and this region is a good target for mutagenic studies to disrupt N protein self-association and virion assembly.Coronaviruses are responsible for ϳ30% of human upper respiratory infections each year. In November 2002, a new coronavirus, known as the severe acute respiratory syndrome (SARS) 1 -associated coronavirus, SARS-CoV, emerged in China and caused more than 8000 cases of SARS worldwide. Approximately 10% of these cases were fatal. Similar to other coronaviruses, SARS-CoV is an enveloped, single-stranded (ss) RNA virus. The SARS-CoV genome contains ϳ29,700 nucleotides (1, 2), encoding the RNA-dependent RNA polymerase and four structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N). In addition, intergenic regions encode several open reading frames for nonstructural proteins of unknown function (1, 2). The S protein is a surface glycoprotein that mediates viral entry by binding to the cellular receptor angiotensin-converting enzyme 2 (ACE2) (3) and inducing membrane fusion. The receptorbinding domain has been mapped to amino acids 318 -510 (4, 5), and structures of the heptad repeat region of S protein (6, 7) indicate that it is a class I membrane fusion protein. The M protein of coronavirus is the most abundant protein component of the envelope. This protein plays a predominant role in the formation and release of the virion envelope. When co-expressed with the E protein, virus-like particles with sizes and shapes similar to those of virions are assembled (8, 9). Recently, virus-like particles of SARS-CoV were obtained by recombinant expression of S, E, and M proteins in insect cells (10). Inside the envelope, the N protein associates with the genomic RNA to form a long, flexible, helical ribonucleoprotein. The N protein is typically 350 -450 amino acids in length, highly basic, and serine-phosphorylated, but the extent and physiological relevance of phosphorylation is unclear (11, 12). In addition to its structural role, several additional functions are postulated for the N protei...

To evaluate the ability of lactate dehydrogenase to facilitate the bond making/breaking steps for both the addition of pyruvate enol to NAD (pyruvate adduct reaction) and the normal redox reaction, the ability of the enzyme to facilitate the tautomerization of bound pyruvate is assessed. In addition, the equilibrium constants for the adduct reaction are obtained for both bound and free reactants from the ratio of the rate constants in the forward and reverse reactions (at pH 7). The latter comparison indicates that the enzyme facilitates bond making/breaking in the (forward) pyruvate adduct reaction by a factor of about 10(11) M. Similar comparisons suggest that reactant immobilization accounts for about 1000 M of this 10(11) M rate effect. Since the (pH-independent) rate constant for the ketonization of bound pyruvate enol assisted by the external buffer, imidazolium ion, is 2 X 10(7) M-1 s-1 and the corresponding rate constant for free pyruvate enol, again assisted by imidazolium ion, is 35 M-1 s-1 [Burger, J. W., II, & Ray, W. J., Jr. (1978) Biochemistry 17, 1664], the enzyme facilitates the bond making/breaking steps associated with the conversion of bound HO-C less than to bound O = C less than by a factor of about 10(6)-fold. The product of the above two rate enhancement factors and the rate factor suggested previously for the environmental effect on NAD produced by its binding to lactate dehydrogenase, 100-fold, is 10(11) M, and it accounts for the bond making/breaking effects exerted by the enzyme in the pyruvate adduct reaction. The rate constant for oxidation of ethanol (a model for lactate) by 1-methylnicotinamide (a model for NAD) is about 5 X 10(-12) M-1 s-1 at 25 degrees C in pure ethanol (delta H for this reaction is about 30 kcal/mol). The ratio of the rate constants for E X NAD X Lac----E X NADH X Pyr and the above model reaction is estimated as about 10(14) M in water; i.e., the LDH-induced rate effect is about 10(14) M. The product of the values for the above rate factors for the normal redox reaction is about 10(12) M. Although the value of this product is less certain than that for the adduct reaction, these rate factors do account for much of the LDH-induced rate effect.

Pyridoxal 5-phosphate (PLP, vitamin B 6 ), a cofactor in many enzymatic reactions, has two distinct biosynthetic routes, which do not coexist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea, and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT produces ammonia from glutamine and PdxS combines ammonia with five-and threecarbon phosphosugars to form PLP. In the 2.2-Å crystal structure, PdxS is a cylindrical dodecamer of subunits having the classic (␤/␣) 8 barrel fold. PdxS subunits form two hexameric rings with the active sites positioned on the inside. The hexamer and dodecamer forms coexist in solution. A novel phosphate-binding site is suggested by bound sulfate. The sulfate and another bound molecule, methyl pentanediol, were used to model the substrate ribulose 5-phosphate, and to propose catalytic roles for residues in the active site. The distribution of conserved surfaces in the PdxS dodecamer was used to predict a docking site for the glutaminase partner, PdxT.

The Syk protein-tyrosine kinase plays a major role in signaling through the B cell receptor for antigen (BCR). Syk binds the receptor via its tandem pair of SH2 domains interacting with a doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the BCR complex. Upon phosphorylation of Tyr-130, which lies between the two SH2 domains distant to the phosphotyrosine binding sites, Syk dissociates from the receptor. To understand the structural basis for this dissociation, we investigated the structural and dynamic characteristics of the wild type tandem SH2 region (tSH2) and a variant tandem SH2 region (tSH2 pm) with Tyr-130 substituted by Glu to permanently introduce a negative charge at this position. NMR heteronuclear relaxation experiments, residual dipolar coupling measurements and analytical ultracentrifugation revealed substantial differences in the hydrodynamic behavior of tSH2 and tSH2 pm. Although the two SH2 domains in tSH2 are tightly associated, the two domains in tSH2 pm are partly uncoupled and tumble in solution with a faster correlation time. In addition, the equilibrium dissociation constant for the binding of tSH2pm to dp-ITAM (1.8 M) is significantly higher than that for the interaction between dp-ITAM and tSH2 but is close to that for a singly tyrosine-phosphorylated peptide binding to a single SH2 domain. Experimental data and hydrodynamic calculations both suggest a loss of domain-domain contacts and change in relative orientation upon the introduction of a negative charge on residue 130. A long-distance structural mechanism by which the phosphorylation of Y130 negatively regulates the interaction of Syk with immune receptors is proposed.allosteric regulation ͉ multidomain dynamics ͉ Syk kinase regulation ͉ tyrosine phosphorylation ͉ NMR 15 N relaxation S yk, a 72-kDa cytoplasmic protein-tyrosine kinase essential to receptor-mediated signaling in B cells, has two N-terminal SH2 domains connected by a 45-residue region (interdomain A). These tandem SH2 domains are separated by a longer, 104-residue region (interdomain B) from a C-terminal kinase domain. Syk mediates B cell signaling through a variety of immune-recognition receptors, including the B cell antigen receptor (BCR). The receptors have similar subunits bearing cytoplasmic, immunoreceptor tyrosinebased activation motifs (ITAMs) (1) with the consensus sequence YXX(L/I)-X 6-9 -YXX(L/I) (2). Phosphorylation on both ITAM tyrosine residues occurs after receptor engagement and is required for Syk binding via the tandem SH2 domains to initiate subsequent signaling cascades (3). Zap-70, the other member of the Syk family, functions similarly in T cell receptor signaling as Syk does in B cells. Events of receptor-dependent signaling in these two cells are highly analogous.Syk is physically and functionally coupled to receptors with ITAMs that vary in the sequence and length of the spacer that separates the two phosphotyrosines (4). The tandem SH2 domains associate with high affinity in a head-to-tail orientation with ea...