Tertiary structural similarity between a class A beta-lactamase and a penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase

beta-Lactam antibiotics--the penicillins, cephalosporins and related compounds--act by inhibiting enzymes that catalyse the final stages of the synthesis of bacterial cell walls. Recent crystallographic studies of representative enzymes are beginning to reveal the structural bases of antibiotic specificity and mechanism of action, while intensive efforts are being made to understand the beta-lactamase enzymes that are largely responsible for bacterial resistance to these antibiotics. It has been suggested that the beta-lactamases and beta-lactam target enzymes may be evolutionarily related and some similarity of amino-acid sequence around a common active-site serine residue supports this idea. We present here the first evidence from a comparison of three-dimensional structures in support of this hypothesis: the structure of beta-lactamase I from Bacillus cereus is similar to that of the penicillin-sensitive D-alanyl-D-alanine carboxypeptidase-transpeptidase from Streptomyces R61.     

Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme

The biosynthetic pathway of the clinically important antibiotic fosfomycin uses enzymes that catalyse reactions without precedent in biology. Among these is hydroxypropylphosphonic acid epoxidase, which represents a new subfamily of non-haem mononuclear iron enzymes. Here we present six X-ray structures of this enzyme: the apoenzyme at 2.0 A resolution; a native Fe(II)-bound form at 2.4 A resolution; a tris(hydroxymethyl)aminomethane-Co(II)-enzyme complex structure at 1.8 A resolution; a substrate-Co(II)-enzyme complex structure at 2.5 A resolution; and two substrate-Fe(II)-enzyme complexes at 2.1 and 2.3 A resolution. These structural data lead us to suggest how this enzyme is able to recognize and respond to its substrate with a conformational change that protects the radical-based intermediates formed during catalysis. Comparisons with other family members suggest why substrate binding is able to prime iron for dioxygen binding in the absence of alpha-ketoglutarate (a co-substrate required by many mononuclear iron enzymes), and how the unique epoxidation reaction of hydroxypropylphosphonic acid epoxidase may occur.     

High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus

Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.     

Novel NADPH-binding domain revealed by the crystal structure of aldose reductase.

Aldose reductase is the first enzyme in the polyol pathway and catalyses the NADPH-dependent reduction of D-glucose to D-sorbitol. Under normal physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. Here we present the crystal structure of pig lens aldose reductase refined to an R-factor of 0.232 at 2.5-A resolution. It exhibits a single domain folded in an eight-stranded parallel alpha/beta barrel, similar to that in triose phosphate isomerase and a score of other enzymes. Hence, aldose reductase does not possess the expected canonical dinucleotide-binding domain. Crystallographic analysis of the binding of 2'-monophospho-adenosine-5'-diphosphoribose, which competitively inhibits NADPH binding reveals that it binds into a cleft located at the C-terminal end of the strands of the alpha/beta barrel. This represents a new type of binding for nicotinamide adenine dinucleotide coenzymes.     

A serine protease triad forms the catalytic centre of a triacylglycerol lipase

True lipases attach triacylglycerols and act at an oil-water interface; they constitute a ubiquitous group of enzymes catalysing a wide variety of reactions, many with industrial potential. But so far the three-dimensional structure has not been reported for any lipase. Here we report the X-ray structure of the Mucor miehei triglyceride lipase and describe the atomic model obtained at 3.1 A resolution and refined to 1.9 A resolution. It reveals a Ser..His..Asp trypsin-like catalytic triad with an active serine buried under a short helical fragment of a long surface loop.     

Tertiary structural differences between microbial serine proteases and pancreatic serine enzymes.

Although primary structural homology between bacterial serine proteases and those from the mammalian pancreas is slight, two-thirds of the residues in the bacterial enzyme SGPB as seen at 2.8-A resolution, adopt a similar polypeptide chain conformation to that of the chymotrypsin family. The three major regions of difference show how this family of proteolytic enzymes has developed from the more primitive bacterial to the relatively sophisticated pancreatic enzymes.     

A voltage-gated ion channel model inferred from the crystal structure of alamethicin at 1.5-A resolution

The crystal structure of alamethicin in nonaqueous solvent has been determined, and refined at 1.5-A resolution. The molecular conformation of the three crystallographically independent molecules is largely alpha-helical with a bend in the helix axis at an internal proline residue. The helix structure is highly amphipathic as most of the solvent-accessible polar atoms lie on a narrow strip of surface parallel to the helix axis. Molecular models for the voltage-gated ion channel, with n-fold symmetry and based on the molecular conformations observed in the crystal, are characterized by strong surface complementarity, a hydrophilic interior and a hydrophobic exterior. The channel structures are stabilized by a hydrated annulus of hydrogen-bonded glutamine residues which produce the greatest restriction in the channel diameter.     

Crystallographic study of a beta-lactam inhibitor complex with elastase at 1.84 A resolution

The continuing discovery and development of beta-lactams as antibiotics has had an unparalleled impact on the overall health and well-being of society. Recently, appropriately substituted cephalosporins were shown to be potent inhibitors of elastase, suggesting a novel therapeutic role for the beta-lactams in the control of emphysema and other degenerative diseases. We have now solved and partially refined at atomic resolution the structure of a complex of porcine pancreatic elastase with the time-dependent irreversible inhibitor 3-acetoxymethyl-7-alpha-chloro-3-cephem-4-carboxylate-1,1-dioxide tert-butyl ester (I), the most potent of the beta-lactam elastase inhibitors yet reported. (Porcine pancreatic elastase is a close relative of the desired drug target, human polymorphonuclear leukocyte elastase.) A mechanism of action is presented, based on the structure and on biochemical evidence (T.-Y.L. et al., in preparation), which clarifies the operational similarities and differences between beta-lactam elastase inhibitors and antibiotics. Features of the reaction include the expulsion of a leaving group at the cephalosporin 3' position and the formation of two covalent bonds with the active site of porcine pancreatic elastase at residues Ser 195 and His 57.     

Structure of C3b reveals conformational changes that underlie complement activity

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.     

Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1

The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.     

Binding of a non-beta-lactam antibiotic to penicillin-binding proteins

In the search for new beta-lactam antibiotics of natural origin, the discoveries of cephamycins and sulfazecins (monobactams) were important turning points in that they accelerated many screening efforts aimed at other new compounds. In our target-directed screening for beta-lactam antibiotics using beta-lactam hypersensitive mutants, we have examined Gram-negative bacteria isolated from natural habitats and have recently reported several types of beta-lactam antibiotics such as cephabacins and formadicins. Here we report a novel antibiotic, lactivicin, found using this system. Although lactivicin has various biological activities commonly observed in beta-lactam antibiotics, it does not possess a beta-lactam ring in its molecule, but has the unique structure of a dicyclic dipeptide.     

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.     

A novel calcium binding site in the galactose-binding protein of bacterial transport and chemotaxis

The refined 1.9-A resolution structure of the periplasmic D-galactose-binding protein (GBP) reveals a calcium ion surrounded by seven ligands, all protein oxygen atoms. A nine-residue loop (amino-acid positions 134-142), which is preceded by a beta-turn and followed by a beta-strand, provides five ligands from every second residue. The last two ligands are supplied by the carboxylate group of Glu 205. The entire GBP Ca2+-binding site adopts a conformation very similar to the site in the 'helix-loop-helix' or 'EF-hand' unit commonly found in intracellular calcium-binding proteins, but without the two helices. Structural analyses have also uncovered the sugar-binding site some 30 A from the calcium and a site for interacting with the membrane-bound trg chemotactic signal transducer approximately 45 A from the calcium. Our results show that a common tight calcium binding site of ancient origin can be tethered to different secondary structures. They also provide the first demonstration of a metal-binding site in a protein which is involved in bacterial active transport and chemotaxis.     

Crystal structure of a retroviral protease proves relationship to aspartic protease family

Retroviral gag, pol and env gene products are translated as precursor polyproteins, which are cleaved by virus-encoded proteases to produce the mature proteins found in virions. On the basis of the conserved Asp-Thr/Ser-Gly sequence at the putative protease active sites, and other biochemical evidence, retroviral proteases have been predicted to be in the family of pepsin-like aspartic proteases. It has been suggested that aspartic proteases evolved from a smaller, dimeric ancestral protein, and a recent model of the human immunodeficiency virus (HIV) protease postulated that a symmetric dimer of this enzyme is equivalent to a pepsin-like aspartic protease. We have now determined the crystal structure of Rous sarcoma virus (RSV) protease at 3-A resolution and find it is dimeric and has a structure similar to aspartic proteases. This structure should provide a useful basis for the modelling of the structures of other retroviral proteases, such as that of HIV, and also for the rational design of protease inhibitors as potential antiviral drugs.     

Structure of the Fc fragment of human IgE bound to its high-affinity receptor Fc epsilonRI alpha

The initiation of immunoglobulin-E (IgE)-mediated allergic responses requires the binding of IgE antibody to its high-affinity receptor, Fc epsilonRI. Crosslinking of Fc epsilonRI initiates an intracellular signal transduction cascade that triggers the release of mediators of the allergic response. The interaction of the crystallizable fragment (Fc) of IgE (IgE-Fc) with Fc epsilonRI is a key recognition event of this process and involves the extracellular domains of the Fc epsilonRI alpha-chain. To understand the structural basis for this interaction, we have solved the crystal structure of the human IgE-Fc-Fc epsilonRI alpha complex to 3.5-A resolution. The crystal structure reveals that one receptor binds one dimeric IgE-Fc molecule asymmetrically through interactions at two sites, each involving one C epsilon3 domain of the IgE-Fc. The interaction of one receptor with the IgE-Fc blocks the binding of a second receptor, and features of this interaction are conserved in other members of the Fc receptor family. The structure suggests new approaches to inhibiting the binding of IgE to Fc epsilonRI for the treatment of allergy and asthma.     

Structure of the 30S ribosomal subunit

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.     

无穷维Hamilton算子纯虚谱的扰动

研究了Hilbert空间X⊕X中的无穷维Hamilton算子HC=[A C 0 -A*]和HF=[A F B -A*]的纯虚谱的扰动,其中R(B)是闭的.给定算子A,B,证明了∩C∈S(X)σi(HC)=σiπ(A),∪C∈S(X)σi(HC)=σi(A),∩F∈S(X)σi(HF)=σiπ(APR(B)⊥),∪F∈S(X)σi(HF)=σi(APR(B)⊥),其中σi(T),σiπ(T),PM和S(X)分别表示T的纯虚谱,纯虚近似谱,全空间到M的正交投影和X中的所有自伴算子所成之集.     

Crystal structure of a self-splicing group I intron with both exons

The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here we report the X-ray crystal structure (3.1-A resolution) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asymmetrically positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.     

Resistance to beta-lactam antibiotics by re-modelling the active site of an E. coli penicillin-binding protein

The beta-lactam antibiotics kill bacteria by inhibiting a set of penicillin-binding proteins (PBPs) that catalyse the final stages of peptidoglycan synthesis. In some bacteria the development of intrinsic resistance to beta-lactam antibiotics by the reduction in the affinity of PBPs causes serious clinical problems. The introduction of beta-lactam antibiotics that are resistant to hydrolysis by beta-lactamases may also result in the emergence of intrinsic resistance among the Enterobacteriaceae. The clinical problems that would arise from the emergence of resistant PBPs in enterobacteria have led us to examine the ease with which Escherichia coli can gain resistance to beta-lactams by the production of altered PBPs. The development of resistant PBPs also provides an interesting example of enzyme evolution, since it requires a subtle re-modeling of the enzyme active centre so that it retains affinity for its peptide substrate but excludes the structurally analogous beta-lactam antibiotics. We show here that only four amino-acid substitutions need to be introduced into PBP 3 of E. coli to produce a strain possessing substantial levels of resistance to a wide variety of cephalosporins. We also show that transfer of the gene encoding the resistant PBP 3 from the chromosome to a plasmid could result in the spread of intrinsic resistance not only to other strains of E. coli but also to other enterobacterial species.     

Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous

Mandelate racemase (MR) and muconate lactonizing enzyme (MLE) catalyse separate and mechanistically distinct reactions necessary for the catabolism of aromatic acids by Pseudomonas putida. The X-ray crystal structure of MR, solved at 2.5 A resolution, reveals that the secondary, tertiary and quaternary structures of MR and MLE are remarkably similar; also, MR and MLE are about 26% identical in primary structure. However, MR has no detectable MLE activity and vice versa. Thus, MR and MLE constitute the first example of enzymes that catalyse different reactions, as opposed to mechanistically identical reactions on different substrates, yet possess sufficient structural and sequence identity that they are likely to have evolved from a common ancestor. The discovery that MR and MLE catalyse different reactions but share a common structural framework has broad implications for the natural evolution of enzymes and metabolic pathways, as well as for the rational modification of enzyme activities.