Vps4 substrate binding and coupled mechanisms of Vps4p substrate recruitment and release from autoinhibition.

The ESCRT pathway's AAA+ ATPase, Vps4p, remodels ESCRT-III complexes to drive membrane fission. Here, we use peptide binding assays to further the understanding of substrate specificity and the mechanism of autoinhibition. Our results reveal unexpected sequence preference to the substrate binding groove and an elegant mechanism of regulation that couples localization to substrate with release from autoinhibition.

Structural and kinetic characterization of mutant human uroporphyrinogen decarboxylases.

Porphyria cutanea tarda (PCT) is caused by inhibition of uroporphyrinogen decarboxylase (URO-D) activity in hepatocytes. Subnormal URO-D activity results in accumulation and urinary excretion of uroporphyrin and heptacarboxyl porphyrin. Heterozygosity for mutations in the URO-D gene is found in the familial form of PCT (F-PCT). Over 70 mutations of URO-D have been described but very few have been characterized structurally. Here we characterize 3 mutations in the URO-D gene found in patients with F-PCT, G318R, K297N, and D306Y. Expression of the D306Y mutation results in an insoluble recombinant protein. G318R and K297N have little effect on the structure or activity of recombinant URO-D, but the proteins display reduced stability in vitro.

Detergent-assisted oxidative folding of delta-conotoxins.

Conotoxins comprise a diverse group of disulfide-rich peptides found in venoms of predatory Conus species. The native conformation of these peptides is marginally stable in comparison with alternative conformations, often resulting in low folding yields. The oxidative folding of hydrophobic delta-conotoxins was found to produce less than 1% of the native peptide [Bulaj, G. et al. (2001) Biochemistry 40, 13201]. In order to identify factors that might improve folding yields, we screened a number of additives including water-soluble polymers, detergents and osmolytes for their ability to increase steady-state accumulation of the native delta-conotoxin PVIA. The presence of a non-ionic detergent Tween and low temperature appeared to be the most effective factors in improving the oxidative folding. The detergent was also effective in promoting folding of other hydrophobic delta-conotoxins. Based on our findings, we discuss a possible mechanism for detergent-assisted folding and the general applicability of this mechanism to facilitating the proper folding of hydrophobic, cysteine-rich peptides.

The 11S regulators of 20S proteasome activity.

Although substantial progress has been made in understanding the biochemical properties of 11S regulators since their discovery in 1992, we still only have a rudimentary understanding of their biological role. As discussed above, we have proposed a model in which the alpha/beta complex promotes the production of antigenic peptides by opening the exit port of the 20S proteasome (Whitby et al. 2000). There are other possibilities, however, that are not exclusive of the exit port hypothesis. For example the alpha/beta complex may promote assembly of immunoproteasome as suggested by Preckel et al. 1999, or it may function as a docking module and conduit for the delivery of peptides to the ER lumen (Realini et al. 1994b). There are also unanswered structural and mechanistic questions. Higher resolution data are needed to discern important structural details of the PA26/20S proteasome complex. The models for binding and activation that are suggested from the structural data have to be tested by mutagenesis and biochemical analysis. What is the role of homolog-specific inserts? Will cognate regulator/proteasome complexes show conformational changes that are not apparent in the currently available crystal structures, including perhaps signs of allosteric communication between the regulator and the proteasome active sites?

Crystal structure of human uroporphyrinogen III synthase.

Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and vitamin B(12). U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two alpha/beta domains connected by a beta-ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.

Functional consequences of naturally occurring mutations in human uroporphyrinogen decarboxylase.

Functional consequences of 12 mutations-10 missense, 1 splicing defect, and 1 frameshift mutation-were characterized in the uroporphyrinogen decarboxylase (URO-D) gene found in Utah pedigrees with familial porphyria cutanea tarda (F-PCT). All but one mutation altered a restriction site in the URO-D gene, permitting identification of affected relatives using a combination of polymerase chain reaction and restriction enzyme digestion. In a bacterial expression system, 3 of the missense mutants were found in inclusion bodies, but 7 were expressed as soluble proteins. Enzymatic activity of soluble, recombinant mutant URO-D genes ranged from 29% to 94% of normal. URO-D mRNA levels in Epstein-Barr-virus transformed cells derived from patients were normal (with the exception of the frameshift mutation) even though protein levels were lower than normal, suggesting that missense mutations generally cause unstable URO-Ds in vivo. The crystal structures of 3 mutant URO-Ds were solved, and the structural consequences of the mutations were defined. All missense mutations reported here and by others were mapped to the crystal structure of URO-D, and structural effects were predicted. These studies define structural and functional consequences of URO-D mutations occurring in patients with F-PCT.

Prolonged gray matter disease without demyelination caused by Theiler's murine encephalomyelitis virus with a mutation in VP2 puff B.

Theiler's murine encephalomyelitis virus (TMEV) is divided into two subgroups based on neurovirulence. During the acute phase, DA virus infects cells in the gray matter of the central nervous system (CNS). Throughout the chronic phase, DA virus infects glial cells in the white matter, causing demyelinating disease. Although GDVII virus also infects neurons in the gray matter, infected mice developed a severe polioencephalomyelitis, and no virus is detected in the white matter or other areas in the CNS in rare survivors. Several sequence differences between the two viruses are located in VP2 puff B and VP1 loop II, which are located near each other, close to the proposed receptor binding site. We constructed a DA virus mutant, DApBL2M, which has the VP1 loop II of GDVII virus and a mutation at position 171 in VP2 puff B. While DApBL2M virus replicated less efficiently than DA virus during the acute phase, DApBL2M-induced acute polioencephalitis was comparable to that in DA virus infection. Interestingly, during the chronic phase, DApBL2M caused prolonged gray matter disease in the brain without white matter involvement in the spinal cord. This is opposite what is observed during wild-type DA virus infection. Our study is the first to demonstrate that conformational differences via interaction of VP2 puff B and VP1 loop II between GDVII and DA viruses can play an important role in making the transition of infection from the gray matter in the brain to the spinal cord white matter during TMEV infection.

Sterols in blood of normal and Smith-Lemli-Opitz subjects.

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag(+)-HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C(27) sterols observed in the SLOS blood samples were cholesterol (12;-98%), 7-dehydrocholesterol (0.4;-44%), 8-dehydrocholesterol (0.5;-22%), and cholesta-5,7,9(11)-trien-3beta-ol (0.02;-5%), whereas the normal blood samples contained <0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05;-0.6%) and cholestanol (0.1;-0.4%) and approximately 0.003;-0.1% each of the Delta(8), Delta(8(14)), Delta(5,8(14)), Delta(5,24), Delta(6,8), Delta(6,8(14)), and Delta(7,24) sterols. The results are consistent with the hypothesis that the Delta(8(14)) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the Delta(5,7,9(11)) sterol. 19-Norcholesta-5,7,9-trien-3beta-ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.

Mutations that alter the surface charge of alpha-tropomyosin are associated with dilated cardiomyopathy.

Proteins in cardiac myocytes assemble into contractile units known as sarcomeres. Contractile force is generated by interaction between sarcomeric thick and thin filaments. Thin filaments also transmit force within and between myocytes. Mutations in genes encoding the thin filament proteins actin and tropomyosin cause hypertrophic cardiomyopathy. Mutations affecting functionally distinct domains of actin also cause dilated cardiomyopathy (DCM). We used a non-positional candidate gene approach to test further the hypothesis that dysfunction of sarcomeric thin filaments, due to different mutations in the same gene, can lead to either hypertrophic or dilated cardiomyopathy. Mutational analyses of alpha-tropomyosin 1 were performed in patients with idiopathic DCM. We identified two mutations that alter highly conserved residues and that, unlike hypertrophic cardiomyopathy-associated mutations, cause localized charge reversal on the surface of tropomyosin. Therefore, substitution of different amino acid residues in the same thin filament proteins is associated with the distinct phenotypes of cardiac hypertrophy or congestive heart failure.

Structural basis for the activation of 20S proteasomes by 11S regulators.

Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a nonspecific and abundant barrel-shaped complex called the 20S proteasome. Substrates access the active sites, which are sequestered in an internal chamber, by traversing a narrow opening (alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences of alpha-subunits. Peptide products probably exit the 20S proteasome through the same opening. 11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are heptamers that stimulate 20S proteasome peptidase activity in vitro and may facilitate product release in vivo. Here we report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma brucei (PA26). PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the 20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that open the gate separating the proteasome interior from the intracellular environment. The reduction in processivity expected for an open conformation of the exit gate may explain the role of 11S regulators in the production of ligands for major histocompatibility complex class I molecules.

Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy.

Mutations in genes encoding sarcomeric proteins cause hypertrophic cardiomyopathy (HCM). The sarcomeric protein actin plays a central, dual role in cardiac myocytes, generating contractile force by interacting with myosin and also transmitting force within and between cells. Two missense mutations in the cardiac actin gene (ACTC), postulated to impair force transmission, have been associated with familial dilated cardiomyopathy (DCM). Recently, a missense mutation in ACTC was found to cosegregate with familial HCM. To further test the hypothesis that mutations within functionally distinct domains of ACTC cause either DCM or HCM, we performed mutational analyses in 368 unrelated patients with familial or sporadic HCM. Single strand conformation polymorphism and sequence analyses of genomic DNA were performed. De novo mutations in ACTC were identified in two patients with sporadic HCM who presented with syncope in early childhood. Patients were heterozygous for missense mutations resulting in Pro164Ala and Ala331Pro amino acid substitutions, adjacent to regions of actin-actin and actin-myosin interaction, respectively. A mutation that cosegregated with familial HCM was also found, causing a Glu99Lys substitution in a weak actomyosin binding domain. The cardiac phenotype in many affected patients was characterized by an apical form of HCM. These findings support the hypothesis that a single amino acid substitution in actin causes either congestive heart failure or maladaptive cardiac hypertrophy, depending on its effect on actin structure and function.

Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains.

This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme FOK:I. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by approximately 4 bp with a 5' overhang, as for native FOK:I. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.

Crystal structure of tropomyosin at 7 Angstroms resolution.

Tropomyosin is a 400A-long coiled coil that polymerizes to form a continuous filament that associates with actin in muscle and numerous non-muscle cells. Tropomyosin and troponin together form a calcium-sensitive switch that is responsible for thin-filament regulation of striated muscle. Subtle structural features of the molecule, including non-canonical aspects of its coiled-coil motif, undoubtedly influence its association with f-actin and its role in thin filament regulation. Previously, careful inspection of native diffraction intensities was sufficient to construct a model of tropomyosin at 9A resolution in a spermine-induced crystal form that diffracts anisotropically to 4A resolution. Single isomorphous replacement (SIR) phasing has now provided an empirical determination of the structure at 7A resolution. A novel method of heavy-atom analysis was used to overcome difficulties in interpretation of extremely anisotropic diffraction. The packing arrangement of the molecules in the crystal, and important aspects of the tropomyosin geometry such as non-uniformities of the pitch and variable bending and radius of the coiled coil are evident.

Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.

Theiler's viruses with mutations in loop I of VP1 lead to altered tropism and pathogenesis.

Theiler's murine encephalomyelitis viruses are picornaviruses that can infect the central nervous system. The DA strain produces an acute polioencephalomyelitis followed by a chronic demyelinating disease in its natural host, the mouse. The ability of DA virus to induce a demyelinating disease renders this virus infection a model for human demyelinating diseases such as multiple sclerosis. Here we describe the generation and characterization of DA virus mutants that contain specific mutations in the viral capsid protein VP1 at sites believed to be important contact regions for the cellular receptor(s). A mutant virus with a threonine-to-aspartate (T81D) substitution in VP1 loop I adjacent to the putative virus receptor binding site exhibited a large-plaque phenotype but had a slower replication cycle in vitro. When this mutant virus was injected into susceptible mice, an altered tropism was seen during the acute stage of the disease and the chronic demyelinating disease was not produced. A virus with a threonine-to-valine substitution (T81V) did not cause any changes in the pattern or extent of disease seen in mice, whereas a virus with a tryptophan substitution at this position (T81W) produced a similar acute disease but was attenuated for the development of the chronic disease. A change in amino acids in a hydrophobic patch located in the wall of the pit, VP1 position 91, to a hydrophilic threonine (V91T) resulted in a profound attenuation of the acute and chronic disease without persistence of virus. This report illustrates the importance of the loop I of VP1 and a site in the wall of the pit in pathogenesis and that amino acid substitutions at these sites result in altered virus-host interactions.

Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes.

The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.

Replacement of loop II of VP1 of the DA strain with loop II of the GDVII strain of Theiler's murine encephalomyelitis virus alters neurovirulence, viral persistence, and demyelination.

Theiler's murine encephalomyelitis viruses, which are murine picornaviruses, can cause central nervous system inflammatory disease. To study the role of loop II in capsid protein VP1, two mutant viruses of strain DA in which DA loop II amino acids were replaced with strain GDVII amino acids were constructed. Infection of mice with the two mutant viruses led to dramatically different patterns of disease.

Crystal structure of human uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. This activity is essential in all organisms, and subnormal activity of URO-D leads to the most common form of porphyria in humans, porphyria cutanea tarda (PCT). We have determined the crystal structure of recombinant human URO-D at 1.60 A resolution. The 40.8 kDa protein is comprised of a single domain containing a (beta/alpha)8-barrel with a deep active site cleft formed by loops at the C-terminal ends of the barrel strands. Many conserved residues cluster at this cleft, including the invariant side chains of Arg37, Arg41 and His339, which probably function in substrate binding, and Asp86, Tyr164 and Ser219, which may function in either binding or catalysis. URO-D is a dimer in solution (Kd = 0.1 microM), and this dimer also appears to be formed in the crystal. Assembly of the dimer juxtaposes the active site clefts of the monomers, suggesting a functionally important interaction between the catalytic centers.

Structure of the proteasome activator REGalpha (PA28alpha).

The specificity of the 20S proteasome, which degrades many intracellular proteins, is regulated by protein complexes that bind to one or both ends of the cylindrical proteasome structure. One of these regulatory complexes, the 11S regulator (known as REG or PA28), stimulates proteasome peptidase activity and enhances the production of antigenic peptides for presentation by class I molecules of the major histocompatibility complex (MHC). The three REG subunits that have been identified, REGalpha, REGbeta and REGgamma (also known as the Ki antigen), share extensive sequence similarity, apart from a highly variable internal segment of 17-34 residues which may confer subunit-specific properties. REGalpha and REGbeta preferentially form a heteromeric complex, although purified REGalpha forms a heptamer in solution and has biochemical properties similar to the heteromeric REGalpha/REGbeta complex. We have now determined the crystal structure of human recombinant REGalpha at 2.8 A resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 A diameter at one end and another of 30 A diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome alpha-subunits through which substrates and products can pass.

Entamoeba histolytica: computer-assisted modeling of phosphofructokinase for the prediction of broad-spectrum antiparasitic agents.

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) is the rate-limiting glycolytic enzyme found in the pathogenic protists Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis, and Naegleria fowleri. The enzyme differs significantly from ATP-dependent phosphofructokinases found in humans and as such represents an important drug target. Current therapy for infections caused by these pathogens is inadequate, especially for children, pregnant women, and the immune compromised. The development of more selective, safer agents in imperative, as parasitic infections are currently a significant health threat worldwide and will likely become increasingly common agents of disease in the future. For the purpose of designing drugs to treat parasitic infections, we have constructed a model of PPi-PFK from E. histolytica based on the three-dimensional structure of the ATP-dependent PFK from Bacillus stearothermophilus. The model was used with the computer program Dock 3.5 (University of California, San Francisco) to predict the binding of pyrophosphate and selected bisphosphonates to the enzyme. The predicted drug-enzyme interactions suggested that two of these compounds would be competitive inhibitors of pyrophosphate. These drugs were tested against E. histolytica and inhibited the growth of amebae in vitro. This class of compounds may have broad-spectrum antiparasitic activity and, in the future, may facilitate the treatment of serious parasitic infections.