Applications of analytical ultracentrifugation to protein size-and-shape distribution and structure-and-function analyses.

The rebirth of modern analytical ultracentrifugation (AUC) began in 1990s. Since then many advanced AUC detectors have been developed that provide a vast range of versatile choices when characterizing the physical and chemical features of macromolecules. In addition, there have been remarkable advances in software that allow the analysis of AUC data using more sophisticated models, including quaternary structures, conformational changes, and biomolecular interactions. Here we report the application of AUC to protein size-and-shape distribution analysis and structure-and-function analysis in the presence of ligands or lipids. Using band-sedimentation velocity, quaternary structural changes and an enzyme's catalytic activity can be observed simultaneously. This provides direct insights into the correlation between quaternary structure and catalytic activity of the enzyme. On the other hand, also in this study, we have applied size-and-shape distribution analysis to a lipid-binding protein in either an aqueous or lipid environment. The sedimentation velocity data for the protein with or without lipid were evaluated using the c(s,f(r)) two-dimensional distribution model, which provides a precise and quantitative means of analyzing the protein's conformational changes.

Mutation of Glu-166 blocks the substrate-induced dimerization of SARS coronavirus main protease.

The maturation of SARS coronavirus involves the autocleavage of polyproteins 1a and 1ab by the main protease (Mpro) and a papain-like protease; these represent attractive targets for the development of anti-SARS drugs. The functional unit of Mpro is a homodimer, and each subunit has a His-41cdots, three dots, centeredCys-145 catalytic dyad. Current thinking in this area is that Mpro dimerization is essential for catalysis, although the influence of the substrate binding on the dimer formation has never been explored. Here, we delineate the contributions of the peptide substrate to Mpro dimerization. Enzyme kinetic assays indicate that the monomeric mutant R298A/L exhibits lower activity but in a cooperative manner. Analytical ultracentrifugation analyses indicate that in the presence of substrates, the major species of R298A/L shows a significant size shift toward the dimeric form and the monomer-dimer dissociation constant of R298A/L decreases by 12- to 17-fold, approaching that for wild-type. Furthermore, this substrate-induced dimerization was found to be reversible after substrates were removed. Based on the crystal structures, a key residue, Glu-166, which is responsible for recognizing the Gln-P1 of the substrate and binding to Ser-1 of another protomer, will interact with Asn-142 and block the S1 subsite entrance in the monomer. Our studies indicate that mutation of Glu-166 in the R298A mutant indeed blocks the substrate-induced dimerization. This demonstrates that Glu-166 plays a pivotal role in connecting the substrate binding site with the dimer interface. We conclude that protein-ligand and protein-protein interactions are closely correlated in Mpro.

Biophysical characterization of a recombinant leucyl aminopeptidase from Bacillus kaustophilus.

The biophysical properties of Bacillus kaustophilus leucyl aminopeptidase (BkLAP) were examined in terms of analytical ultracentrifugation, fluorescence spectroscopy, and circular dichroism. By using the analytical ultracentrifuge, we demonstrated that tetrameric BkLAP exists as the major form in solution at protein concentration of 1.5 mg/ml at pH 8.0. The native enzyme started to unfold beyond ~1 M GdnHCl and reached an unfolded intermediate with [GdnHCl](1/2) at 1.8 M. Thermal unfolding of BkLAP was found to be highly irreversible and led to a marked formation of aggregates.

Thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deISGylating enzyme.

In the search for effective therapeutics against severe acute respiratory syndrome (SARS), 6-mercaptopurine (6MP) and 6-thioguanine (6TG) were found to be specific inhibitors for the SARS-coronavirus (CoV) papain-like protease (PLpro), a cysteine protease with deubiquitinating and deISGylating activity. 6MP and 6TG have long been used in cancer chemotherapy for treatment of acute lymphoblastic or myeloblastic leukaemia. Development and optimization of 6MP and 6TG will not only be important for antiviral studies, but also for further elucidating the biological functions of cellular deubiquitinating enzymes (DUBs) and deISGylating enzymes. So far, several crystal structures of cellular DUBs have been solved. Structure comparison has been carried out to search for DUBs with a similar structure to that of PLpro, and we have tried to dock 6MP and 6TG into these DUBs to investigate the potential use of 6MP and 6TG as cellular DUB inhibitors. The best docking score and binding energy for 6MP and 6TG is against ubiquitin-specific protease (USP)14, suggesting that 6MP and 6TG are potential inhibitors of USP14. Finding new usages for old drugs will speed up the process of drug discovery and substantially reduce the cost of drug development.

The roles of Tyr(91) and Lys(162) in general acid-base catalysis in the pigeon NADP+-dependent malic enzyme.

The role of general acid-base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr(91) and Lys(162) residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in k(cat) values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the k(cat) values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(k(cat)) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pK(a) value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys(162) as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr(91) residue could form a hydrogen bond with Lys(162) to act as a catalytic dyad that contributes a proton to complete the enol-keto tautomerization.

Correlation between dissociation and catalysis of SARS-CoV main protease.

The dimeric interface of severe acute respiratory syndrome coronavirus main protease is a potential target for the anti-SARS drug development. We have generated C-terminal truncated mutants by serial truncations. The quaternary structure of the enzyme was analyzed using both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Global analysis of the combined results showed that truncation of C-terminus from 306 to 300 had no appreciable effect on the quaternary structure, and the enzyme remained catalytically active. However, further deletion of Gln-299 or Arg-298 drastically decreased the enzyme activity to 1-2% of wild type (WT), and the major form was a monomeric one. Detailed analysis of the point mutants of these two amino acid residues and their nearby hydrogen bond partner Ser-123 and Ser-139 revealed a strong correlation between the enzyme activity loss and dimer dissociation.

Metal ions stabilize a dimeric molten globule state between the open and closed forms of malic enzyme.

Malic enzyme is a tetrameric protein with double dimer quaternary structure. In 3-5 M urea, the pigeon cytosolic NADP(+)-dependent malic enzyme unfolded and aggregated into various forms with dimers as the basic unit. Under the same denaturing conditions but in the presence of 4 mM Mn(2+), the enzyme existed exclusively as a molten globule dimer in solution. Similar to pigeon enzyme (Chang, G. G., T. M. Huang, and T. C. Chang. 1988. Biochem. J. 254:123-130), the human mitochondrial NAD(+)-dependent malic enzyme also underwent a reversible tetramer-dimer-monomer quaternary structural change in an acidic pH environment, which resulted in a molten globule state that is also prone to aggregate. The aggregation of pigeon enzyme was attributable to Trp-572 side chain. Mutation of Trp-572 to Phe, His, Ile, Ser, or Ala abolished the protective effect of the metal ions. The cytosolic malic enzyme was completely digested within 2 h by trypsin. In the presence of Mn(2+), a specific cutting site in the Lys-352-Gly-Arg-354 region was able to generate a unique polypeptide with M(r) of 37 kDa, and this polypeptide was resistant to further digestion. These results indicate that, during the catalytic process of malic enzyme, binding metal ion induces a conformational change within the enzyme from the open form to an intermediate form, which upon binding of L-malate, transforms further into a catalytically competent closed form.

Structural variation manipulates the differential oxidative susceptibility and conformational stability of apolipoprotein E isoforms.

A growing amount of evidence implicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease (AD). It is now generally believed that the epsilon4 allele is associated with AD and the oxidative stress is more pronounced in AD. However, only limited data are available on apoE isoform-specificity and its relationship to both the oxidative susceptibility and conformational stability of apoE. In this article, we use site-directed mutagenesis to investigate the structural role of amino acid residue 112, which is the only differing residue between apoE3 and E4. We examine the structural variation manipulating the oxidative susceptibility and conformational stability of apolipoprotein E isoforms. Arg112 in apoE4 was changed to Ala and Glu. Previous research has reported that apoE4 is more susceptible to free radicals than apoE3. In protein oxidation experiments, apoE4-R112A becomes more resistant to free radicals to the same extent as apoE3. In contrast, apoE4-R112E becomes the most susceptible protein to free radicals among all the apoE proteins. We also examine the conformational stability and the quaternary structural change by fluorescence spectroscopy and analytical ultracentrifugation, respectively. ApoE3 and E4 show apparent three- and two-state unfolding patterns, respectively. ApoE4-R112A, similar to apoE3, demonstrates a biphasic denaturation with an intermediate that appears. The denaturation curve for apoE4-R112E, however, also displays a biphasic profile but with a slight shoulder at approximately 1.5M GdmCl, implying that an unstable intermediate existed in the denaturation equilibrium. The size distribution of apoE isoforms display similar patterns. ApoE4-R112E, however, has a greater tendency to dissociate from high-molecular-weight species to tetramers. These experimental data suggest that the amino acid residue 112 governs the differences in salt-bridges between these two isoforms and thus has a significant impact on the free radical susceptibility and structural variation of the apoE isoforms.

Critical roles of conserved carboxylic acid residues in pigeon cytosolic NADP+-dependent malic enzyme.

Malic enzyme catalyses the reduction of NADP+ to NADPH and the decarboxylation of L-malate to pyruvate through a general acid/base mechanism. Previous kinetic and structural studies differ in their interpretation of the amino acids responsible for the general acid/base mechanism. To resolve this discrepancy, we used site-directed mutagenesis and kinetic analysis to study four conserved carboxylic amino acids. With the D257A mutant, the Km for Mn2+ and the kcat decreased relative to those of the wild-type by sevenfold and 28-fold, respectively. With the E234A mutant, the Km for Mg2+ and L-malate increased relative to those of the wild-type by 87-fold and 49-fold, respectively, and the kcat remained unaltered, which suggests that the E234 residue plays a critical role in bivalent metal ion binding. The kcat for the D235A and D258A mutants decreased relative to that of the wild-type by 7800-fold and 5200-fold, respectively, for the overall reaction, by 800-fold and 570-fold, respectively, for the pyruvate reduction partial reaction, and by 371-fold and 151-fold, respectively, for the oxaloacetate decarboxylation. The activities of the overall reaction and the pyruvate reduction partial reaction of the D258A mutant were rescued by the presence of 50 mM sodium azide. In contrast, small free acids did not have a rescue effect on the activities of the E234A, D235A, and D257A mutants. These data suggest that D258 may act as a general base to extract the hydrogen of the C2 hydroxy group of L-malate with the aid of D235-chelated Mn2+ to polarize the hydroxyl group.

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme).

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

Characterization of the interactions between Asp141 and Phe236 in the Mn2+-l-malate binding of pigeon liver malic enzyme.

The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+-ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141-->Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (DeltaDeltaG(int)) between any two residues. The dissociation constants for Mn2+ ( K (d,Mn)) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the K (d,Mn) for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of -2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.

Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme.

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity.

Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF-1). SOS is found to bind to nFGF-1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size-exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF-1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n-FGF-1 shows significant cell proliferation activity. (15)N and (1)H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF-1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by beta-strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS-nFGF-1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin-induced oligomerization of nFGF-1 is not mandatory for its cell proliferation activity.

Metal-Induced reversible structural interconversion of human mitochondrial NAD(P)+-dependent malic enzyme.

Human mitochondrial NAD(P)+-dependent malic enzyme was strongly inhibited by Lu3+. The X-ray crystal structures indicated a structural change between the metal-free and Lu3+-containing enzymes (Yang Z, Batra R, Floyd DL, Hung HC, Chang GG, Tong L. Biochem Biophys Res Commun 2000;274:440-444). We characterized the reversible slow-binding mechanism and the structural interconversion between Mn2+- and Lu3+-containing human mitochondrial malic enzymes. When Lu3+ was added, the activity of the human enzyme showed a downward curve over time, similar to that of the pigeon enzyme. The rate of the transformation (k(obs)) from the initial rate to the steady-state rate increased hyperbolically with the concentration of Lu3+, suggesting the involvement of an isomerization step. Lu3+ had a much higher affinity for the isomerized form (K*(i,Lu (app)) = 4.8 microM) than that of the native form (K(i,Lu (app)) = 148 microM). When an excess of Mn2+ was added to the Lu3+-inhibited enzyme, assays of the kinetic activity showed an upward trend, indicating reactivation. This result also indicated that the reactivation was a slow process. Fluorescence quenching experiments confirmed that the Lu3+-induced isomerization was completely reversible. The dynamic quenching constants for the metal-free, Mn2+-containing, and Lu3+-containing enzyme were 3.08, 3.07, and 3.8 M(-1), respectively. When the Lu3+-containing enzyme was treated with excess Mn2+, the dynamic quenching constant returned to the original value (3.09 M(-1)). These results indicated that binding of Mn2+ did not induce any conformational change in the enzyme. The open form transformed to the closed form only after substrate binding. Lu3+, on the other hand, transformed the open form into a catalytically inactive form. Excess Mn2+ could replace Lu3+ in the metal binding site and convert the inactive form back into the open form. This reversible process was slow in both directions because of the same but opposite structural change involved.

Apoptotic effect of caffeic acid phenethyl ester and its ester and amide analogues in human cervical cancer ME180 cells.

BACKGROUND: Caffeic acid phenylether ester (CAPE) has potent antioxidant, anti-inflammatory, antiviral, anti-proliferative, immunomodulatory and pro-apoptotic activities. The activities of CAPE and its novel synthetic derivatives, caffeic acid octyl ester (CAO) and 1-octyl caffeamide (CAN-8), were investigated in this study. MATERIALS AND METHODS: Cultured human cells were incubated with or without these compounds. The effect of these compounds on cell apoptosis, intracellular level of hydrogen peroxide and mitochondrial potential were analyzed. Western blot analysis was used to study the effect of alterations in protein level of caspases, Bcl-2 family, p21, p53 and c-Jun upon drug treatment. RESULTS: These compounds arrested cell proliferation, triggered cell apoptosis and caused a marked scavenging effect of hydrogen peroxide. Apoptosis induced by CAPE or CAO is associated with increased expression of p53, p21 and c-Jun. While the levels of Bcl-2 and Bcl-xL were relatively unchanged, these compounds induced a marked reduction in Mcl-1 level. The CAPE- or CAO-induced apoptosis was also accompanied by a rapid loss of mitochondrial transmembrane potential and activation of caspase-3 and caspase-8, suggesting a mitochondrial-dependent mechanism. In causing these cellular actions, CAO was shown to be comparable or more potent than CAPE, whereas the amide analogue CAN-8 displayed much weaker activities than both CAPE and CAO. Since these three compounds contain similar antioxidant functionality, the difference in their potency suggests that the octyl moiety in CAO is an important determinant for the enhanced activities. CONCLUSION: We have characterized a novel CAPE structure analogue, CAO, which showed strong antioxidant and proapoptotic activities. In addition, we demonstrated that down-regulation of Mcl-1 gene expression and activation of caspase-8 are associated with CAPE-triggered cell apoptosis.

Essential covalent linkage between the chymotrypsin-like domain and the extra domain of the SARS-CoV main protease.

The main protease of the coronavirus causing severe acute respiratory syndrome performs proteolytic processing of the viral polyproteins. The active form of the enzyme is a homodimer with each subunit consisting of three structural domains. Domains I and II, hosting the complete catalytic machinery, constitute the N-terminal chymotrypsin-like folding scaffold and connect to the extra C-terminal domain III by a long loop. Previously, the domain III-truncated enzyme was demonstrated to fold independently into an intact chymotrypsin-like fold, but it showed no enzyme activity. To further delineate the structure-function relationships of the domain III and the long loop, we generated some truncated and mutated M(pro) forms bearing various combinations of the loop with other structural parts of the enzyme. Their conformational and association properties were investigated in detail. Far-ultraviolet circular dichroism (CD) measurements revealed that these fragments could fold independently. The secondary, tertiary and quaternary structures of these mixtures were monitored by CD, fluorescence spectroscopy and analytical ultracentrifugation. However, no enzyme activity was observed for any mutant or mixtures. These observations indicate that the covalent linkage between the chymotrypsin like and the extra domain is essential for enzymatic activity of the main coronavirus protease and for the integrity of its quaternary structure.

Thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus.

The papain-like protease of severe acute respiratory syndrome coronavirus (PLpro) (EC 3.4.22.46) is essential for the viral life cycle and therefore represents an important antiviral target. We have identified 6MP and 6TG as reversible and slow-binding inhibitors of SARS-CoV PLpro, which is the first report about small molecule reversible inhibitors of PLpro. The inhibition mechanism was investigated by kinetic measurements and computer docking. Both compounds are competitive, selective, and reversible inhibitors of the PLpro with K(is) values approximately 10 to 20 microM. A structure-function relationship study has identified the thiocarbonyl moiety of 6MP or 6TG as the active pharmacophore essential for these inhibitions, which has not been reported before. The inhibition is selective because these compounds do not exert significant inhibitory effects against other cysteine proteases, including SARS-CoV 3CLpro and several cathepsins. Thus, our results present the first potential chemical leads against SARS-CoV PLpro, which might be used as lead compounds for further optimization to enhance their potency against SARS-CoV. Both 6MP and 6TG are still used extensively in clinics, especially for children with acute lymphoblastic or myeloblastic leukemia. In light of the possible inhibition against subset of cysteine proteases, our study has emphasized the importance to study in depth these drug actions in vivo.

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride.

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.

Crystal structure of murine CstF-77: dimeric association and implications for polyadenylation of mRNA precursors.

Cleavage stimulation factor (CstF) is a heterotrimeric protein complex essential for polyadenylation of mRNA precursors. The 77 kDa subunit, CstF-77, is known to mediate interactions with the other two subunits of CstF as well as with other components of the polyadenylation machinery. We report here the crystal structure of the HAT (half a TPR) domain of murine CstF-77, as well as its C-terminal subdomain. Structural and biochemical studies show that the HAT domain consists of two subdomains, HAT-N and HAT-C domains, with drastically different orientations of their helical motifs. The structures reveal a highly elongated dimer, spanning 165 A, with the dimerization mediated by the HAT-C domain. Light-scattering studies, yeast two-hybrid assays, and analytical ultracentrifugation measurements confirm this self-association. The mode of dimerization and the relative arrangement of the HAT-N and HAT-C domains are unique to CstF-77. Our data support a role for CstF dimerization in pre-mRNA 3' end processing.

Human spot 14 protein interacts physically and functionally with the thyroid receptor.

Spot 14 (S14) is a small acidic protein with no sequence similarity to other mammalian gene products. Its biochemical function is elusive. Recent studies have shown that, in some cancers, human S14 (hS14) localizes to the nucleus and is amplified, suggesting that it plays a role in the regulation of lipogenic enzymes during tumorigenesis. In this study, we purified untagged hS14 protein and then demonstrated, using various biochemical methods, including analytic ultracentrifugation, that hS14 might form a homodimer. We also found several lines of evidence to suggest physical and functional interactions between hS14 and the thyroid hormone receptor (TR). The ubiquitous expression of hS14 in various cell lines and its cell-type-dependent functions demonstrated in this study suggest that it acts as a positive or negative cofactor of the TR to regulate malic enzyme gene expression. These findings provide a molecular rationale for the role of hS14 in TR-dependent transcriptional activation of the expression of specific genes.