Miroslaw Cygler

Structure of rat procathepsin B: model for inhibition of cysteine protease activity by the proregion

BACKGROUNDnCysteine proteases of the papain superfamily are synthesized as inactive precursors with a 60-110 residue N-terminal prosegment. The propeptides are potent inhibitors of their parent proteases. Although the proregion binding mode has been elucidated for all other protease classes, that of the cysteine proteases remained elusive.nnnRESULTSnWe report the three-dimensional structure of rat procathepsin B, determined at 2.8 A resolution. The 62-residue proregion does not form a globular structure on its own, but folds along the surface of mature cathepsin B. The N-terminal part of the proregion packs against a surface loop, with Trp24p (p indicating the proregion) playing a pivotal role in these interactions. Inhibition occurs by blocking access to the active site: part of the proregion enters the substrate-binding cleft in a similar manner to a natural substrate, but in a reverse orientation.nnnCONCLUSIONSnThe structure of procathepsin B provides the first insight into the mode of interaction between a mature cysteine protease from the papain superfamily and its prosegment. Maturation results in only one loop of cathepsin B changing conformation significantly, replacing contacts lost by removal of the prosegment. Contrary to many other proproteases, no rearrangement of the N terminus occurs following activation. Binding of the prosegment involves interaction with regions of the enzyme remote from the substrate-binding cleft and suggests a novel strategy for inhibitor design. The region of the prosegment where the activating cleavage occurs makes little contact with the enzyme, leading to speculation on the activation mechanism.

Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering

Disulfide bridges were introduced into Cry1Aa, a Bacillus thuringiensis lepidopteran toxin, to stabilize different protein domains including domain I α‐helical regions thought to be involved in membrane integration and permeation. Bridged mutants could not form functional ion channels in lipid bilayers in the oxidized state, but upon reduction with β‐mercaptoethanol, regained parental toxin channel activity. Our results show that unfolding of the protein around a hinge region linking domain I and II is a necessary step for pore formation. They also suggest that membrane insertion of the hydrophobic hairpin made of α‐helices 4 and 5 in domain I plays a critical role in the formation of a functional pore.

Structure and conformational flexibility of Candida rugosa lipase.

Three-dimensional structures of a number of lipases determined in the past decade have provided a solid structural foundation for our understanding of lipase function. The structural studies of Candida rugosa lipase summarized here have addressed many facets of interfacial catalysis. These studies have revealed a fold and catalytic site common to other lipases. Different conformations likely to correlate with interfacial activation of the enzyme were observed in different crystal forms. The structures of enzyme-inhibitor complexes have identified the binding site for the scissile fatty acyl chain, provided the basis for molecular modeling of triglyceride binding and provided insight into the structural basis of the common enantiopreferences shown by lipases.

CRYSTAL STRUCTURE OF PROTEUS VULGARIS CHONDROITIN SULFATE ABC LYASE I AT 1.9A RESOLUTION.

Chondroitin Sulfate ABC lyase I from Proteus vulgaris is an endolytic, broad-specificity glycosaminoglycan lyase, which degrades chondroitin, chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate, and hyaluronan by beta-elimination of 1,4-hexosaminidic bond to unsaturated disaccharides and tetrasaccharides. Its structure revealed three domains. The N-terminal domain has a fold similar to that of carbohydrate-binding domains of xylanases and some lectins, the middle and C-terminal domains are similar to the structures of the two-domain chondroitin lyase AC and bacterial hyaluronidases. Although the middle domain shows a very low level of sequence identity with the catalytic domains of chondroitinase AC and hyaluronidase, the residues implicated in catalysis of the latter enzymes are present in chondroitinase ABC I. The substrate-binding site in chondroitinase ABC I is in a wide-open cleft, consistent with the endolytic action pattern of this enzyme. The tryptophan residues crucial for substrate binding in chondroitinase AC and hyaluronidases are lacking in chondroitinase ABC I. The structure of chondroitinase ABC I provides a framework for probing specific functions of active-site residues for understanding the remarkably broad specificity of this enzyme and perhaps engineering a desired specificity. The electron density map showed clearly that the deposited DNA sequence for residues 495-530 of chondroitin ABC lyase I, the segment containing two putative active-site residues, contains a frame-shift error resulting in an incorrectly translated amino acid sequence.

The Structure of the RlmB 23S rRNA Methyltransferase Reveals a New Methyltransferase Fold with a Unique Knot

In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.

Structural basis for specificity of papain-like cysteine protease proregions toward their cognate enzymes

Synthetic peptides corresponding to the proregions of papain‐like cysteine proteases have been shown to be good and selective inhibitors of their parental enzymes. The molecular basis for their selectivity, quite remarkable in some cases, is not fully understood. The recent determination of the crystal structures of three distinct papain‐like cysteine protease zymogens allows detailed structural comparisons to be made. The reasons for the specificity shown by each proregion toward its cognate enzyme are explained in terms of the three‐dimensional structure of the proregion and the interface between the mature enzyme and the proregion. These comparisons reveal that insertion and substitution of amino acids within the proregion cause major rearrangement of sidechains on the enzyme/proregion interface, allowing detailed surface and charge recognition. Proteins 32:504–514, 1998.

Structure of apoptosis-linked protein ALG-2: insights into Ca2+-induced changes in penta-EF-hand proteins.

BACKGROUNDnThe Ca2+ binding apoptosis-linked gene-2 (ALG-2) protein acts as a proapoptotic factor in a variety of cell lines and is required either downstream or independently of caspases for apoptosis to occur. ALG-2 belongs to the penta-EF-hand (PEF) protein family and has two high-affinity and one low-affinity Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1).nnnRESULTSnWe present the 2.3 A resolution crystal structure of Ca2+-Ioaded des1-20ALG-2 (aa 21-191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight alpha helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different from that of calpain and grancalcin. This difference can be described as a rigid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cleaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule.nnnCONCLUSIONSnA different relative orientation of the N- and C-terminal halves of des1-20ALG-2 in the presence of Ca2+ and the peptide as compared to other Ca2+loaded PEF proteins changes substantially the shape of the molecule, exposing a hydrophobic patch on the surface for peptide binding and a large cleft near the dimer interface. We postulate that the binding of a Gly/ Pro-rich peptide in the presence of Ca2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins.

Enantioselectivity of Candida Rugosa Lipase Toward Carboxylic Acids: A Predictive Rule from Substrate Mapping and X-Ray Crystallography

We used substrate mapping to develop a rule that predicts which enantiomer of chiral carboxylic acid esters reacts faster in hydrolyses catalyzed by lipase from Candida rugosa (CRL, triacylglycerol hydrolase, E. C. 3.1.1.3). This rule, based on the size of the substituents at the stereocenter, is not reliable for crude CRL. It predicts the favoured enantiomer for only 23 out of 34 examples, 68% reliability. However, this rule is completely reliable for purified CRL; it predicts the favoured enantiomer for all 16 examples correctly. The examples include arylpropanoicacids, aryloxypropanoic acids, α-halophenylacetic acids, mandelic acid and O-methylmandelic acid. Further, purified CRL did not catalyse the hydrolysis of N-CBZ-phenylalanine methyl ester and N-CBZ-norleucine methyl ester. These two substrates were exceptions to the rule with crude CRL as the catalyst. Besides eliminating several exceptions, purification also raised the enantioselectivity of CRL toward carboxylic acid esters. To provide a struc...

Recommendations for Nomenclature in Cholinesterases

Information gathered on the genomic organization of cholinesterase genes, advances in biochemical characterization of the various subunits and the elucidation of the tertiary structure of the T. californica enzyme can serve as a basis for a unified nomenclature in cholinesterase research. Herein are some recommendations for nomenclature of catalytic and structural subunits, for designation of cholinesterase gene exons and secondary structure motifs, and for numeration of amino acid positions.

Structure of the noncatalytic domains and global fold of the protein disulfide isomerase ERp72.

Protein disulfide isomerases are a family of proteins that catalyze the oxidation and isomerization of disulfide bonds in newly synthesized proteins in the endoplasmic reticulum. The family includes general enzymes such as PDI that recognize unfolded proteins, and others that are selective for specific classes of proteins. Here, we report the X-ray crystal structure of central non-catalytic domains of a specific isomerase, ERp72 (also called CaBP2 and protein disulfide-isomerase A4) from Rattus norvegicus. The structure reveals strong similarity to ERp57, a PDI-family member that interacts with the lectin-like chaperones calnexin and calreticulin but, unexpectedly, ERp72 does not interact with calnexin as shown by isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. Small-angle X-ray scattering (SAXS) of ERp72 was used to develop models of the full-length protein using both rigid body refinement and ab initio simulated annealing of dummy atoms. The two methods show excellent agreement and define the relative positions of the five thioredoxin-like domains of ERp72 and potential substrate or chaperone binding sites.