Computational Tools For Protein Modeling Pp.1-21
Recent Progress in the Solid-phase Synthesis of Glycopeptide. Pp. 23-48.
Construction of Macromolecular Assemblages in Eukaryotic Processes and their Role in Human Disease: Linking RINGs Together. Pp. 49-73.
A. Kentsis and K. L. B. Borden
Serratia Type Pore Forming Toxins. Pp. 75-89.
Ralf Hertle
The C-Terminal Domain of Pancreatic Lipase: Functional and Structural Analogies with C2 Domains. Pp. 91-103.
Chahinian H.,
Sias B. and Carrière F.
Chitinolytic Enzymes: Catalysis, Substrate Binding, and their Application. Pp105-124.
Tamo Fukamizo
[Back to top] Computational Tools For Protein Modeling
Dong Xu , Ying Xu and Edward C.
Uberbacher
Protein modeling is playing a more and more important role in protein and peptide sciences due to improvements in modeling methods, advances in computer technology, and the huge amount of biological data becoming available. Modeling tools can often predict the structure and shed some light on the function and its underlying mechanism. They can also provide insight to design experiments and suggest possible leads for drug design. This review attempts to provide a comprehensive introduction to major computer programs, especially on-line servers, for protein modeling. The review covers the following aspects: (1) protein sequence comparison, including sequence alignment/search, sequence-based protein family classification, domain parsing, and phylogenetic classification; (2) sequence annotation, including annotation/prediction of hydrophobic profiles, transmembrane regions, active sites, signaling sites, and secondary structures; (3) protein structure analysis, including visualization, geometry analysis, structure comparison/classification, dynamics, and electrostatics; (4) three-dimensional structure prediction, including homology modeling, fold recognition using threading, ab initio prediction, and docking. We will address what a user can expect from the computer tools in terms of their strengths and limitations. We will also discuss the major challenges and the future trends in the field. A collection of the links of tools can be found at http://compbio.ornl.gov/structure/resource/.
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[Back to top] Recent Progress in the Solid-phase Synthesis of Glycopeptide..
Hironobu Hojo and Yoshiaki
Nakahara
The recent understanding of the biological role of glycoproteins has brought about a demand for the highly homogeneous glycopeptides as the functional model for glycoproteins. Thus, much efforts have been made to establish easy and efficient method for glycopeptide synthesis. In this paper, we briefly review the recent advances in the synthesis of O- and N-linked glycopeptide based on the solid-phase method. In O-glycopeptide section, the preparation of glycosylated amino acid units with mucin type and other O-linked carbohydrate chains and their use for solid-phase synthesis are summarized. Other approaches, such as the glycosylation of resin bound peptide are also overviewed. In N-glycopeptide section, the synthesis using glycosylated amino acid units as well as other methods are described..
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[Back to top] Construction of Macromolecular Assemblages in Eukaryotic Processes and their Role in Human Disease: Linking RINGs Together.
A. Kentsis
and K. L. B. Borden
Members
of the Really Interesting New Gene (RING) family of proteins are found
throughout the cells of eukaryotes and function in processes as diverse as
development, oncogenesis, viral replication and apoptosis. There are over 200
members of the RING family where membership is based on the presence of a
consensus sequence of zinc binding residues. Outside of these residues there is
little sequence homology; however,
there are conserved
structural features. Current
evidence strongly suggests that RINGs are protein interaction domains.
We examine the features of RING binding motifs in terms of individual cases and
the potential for finding a universal consensus sequence for RING binding
domains (FRODOs). This review examines known and potential functions of RINGs,
and attempts to develop a framework within which their seemingly multivalent cellular
roles can be consistently understood in their structural and biochemical
context. Interestingly, some RINGs can self-associate as well as bind other
RINGs. The ability to self-associate is typically translated into the annoying
propensity of these domains to aggregate during biochemical characterization.
The RINGs of PML, BRCA1, RAG1, KAP1/TIF1b, Polycomb
proteins, TRAFs and the viral protein Z have been well characterized in terms
of both biochemical studies and functional data and so will serve as focal
points for discussion. We suggest physiological functions for the oligomeric
properties of these domains, such as their role in formation of macromolecular
assemblages which function in an intricate interplay of coupled metal binding,
folding and aggregation, and participate in diverse functions: epigenetic
regulation of gene expression, RNA transport, cell cycle control,
ubiquitination, signal transduction and organelle assembly.
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[Back to top] Serratia Type Pore Forming Toxins..
Ralf Hertle
The Serratia marcescens hemolysin represents a new type of hemolysin and has been studied in great molecular detail with regard to structure, activation and secretion. It has nothing in common with the pore forming toxins of E. coli type (RTX toxins), the Staphylococcus aureus a-toxin or the thiol activated toxin of group A b-hemolytic streptococci (Streptolysin O). Studies on erythrocytes, eukaryotic cells and artificial black lipid membranes, have shown that the mechanism of pore formation of ShlA is different form other pore forming toxins. The S. marcescens hemolysin proteins ShlB and ShlA exhibit protein sequence homologues in Proteus mirabilis, Haemophilus ducreyi, Edwardsiella tarda and Erwinia chrysantemi. Furthermore, sequence motifs present in ShlA and Shlb have been shown to be important for activity and secretion of the S. marcescens hemolysin. Thus, the S. marcescens hemolysin forms the prototype of a new class of hemolysins and of a new secretory mechanism. The uniqueness of this new mechanism is underlined by the fact that activation of ShlA by ShlB strictly requires phosphatidylethanolamine as a cofactor. New data implicate a conformational change in ShlA during activation. In addition, ShlA not only forms pores in erythrocytes but also in fibroblasts and epithelial cells. The cytotoxic action of ShlA is mainly determined by lysis of infected cells in vitro. In sublytic doses, as will normally be the situation in vivo, ShlA exerts additionally effects which are currently under investigation. The knowledge of the structure, activation, secretion and mode of action of S. marcescens hemolysin has implications for proteins, related in sequence or in mode of secretion and activation.
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[Back to top] The C-Terminal Domain of Pancreatic Lipase: Functional and Structural Analogies with C2 Domains..
Chahinian H.,
Sias B. and Carrière F.
The 3D structure of pancreatic lipase (PL)
consists of two functional domains. The N-terminal domain belongs to the a/b
hydrolase fold and contains the active site, which involves a catalytic triad
analogous to that present in serine proteases. The b-sandwich C-terminal domain of PL plays an important part in
the binding process between the lipase and colipase, the specific PL cofactor.
Recent structure-function studies have suggested that the PL C-terminal domain
may have an extra role apart from that of binding colipase. This domain
contains an exposed hydrophobic loop (b5’)
which was found to be located on the same side as the hydrophobic loops
surrounding the active site, and it may be involved in the lipid binding
process. Indirect evidence for this new function of the PL C-terminal domain
has been provided by studies with monoclonal antibodies directed against the b5’ loop. The catalytic activity of the
PL-antibody complexes on water insoluble substrates decreased drastically,
whereas their esterase activity on a soluble substrate remained unchanged.
During the last few years, a number of protein structures (15-lipoxygenase, a-toxin from Clostridium perfringens) have been determined that contain domains
with close structural homologies with the b-sandwich
C-terminal domain of PL. Generally speaking, these domains show structural
homologies with the C2 domains occurring in a wide range of proteins involved
in signal transduction (e.g. phosphoinositide-specific phospholipase C, protein
kinase C, cytosolic phospholipase A2), membrane traffic (e.g. synaptotagmin I,
rabphilin) and membrane disruption (e.g. perforin). Here it is proposed to
review the structure and function of the C2 domains, based on the recent 3D
structures and improved sequence alignments.
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[Back to top] Chitinolytic Enzymes: Catalysis, Substrate Binding, and their Application.
Tamo Fukamizo
After the epoch-making report on X-ray crystal structure of a lysozyme-N-acetylglucosamine trisaccharide complex in 1967, catalytic mechanisms of glycosyl hydrolases have been discussed with reference to the lysozyme mechanism. From the recent findings of chitinolytic enzymes, however, the enzymes were found to have catalytic and substrate binding mechanisms different from those of lysozyme. Based on the X-ray crystal structures of chitinases and their complexes with substrate analogues, the catalytic mechanisms were discussed considering the relative locations of catalytic residues to the bound substrate analogues. Resembling the lysozyme catalytic center, family 19 chitinases, family 46 chitosanases, and family 23 lysozymes have two carboxyl groups at the catalytic center, which are separated (> 10 Å) on either side of the catalytic cleft. The catalytic reaction of the enzymes takes place through a single displacement mechanism. In family 18 chitinases, one can identify only one catalytic carboxylate as a proton donor, but not the second catalytic carboxylate whose function and location are similar to those of Asp52 in lysozyme. The catalytic reaction of family 18 chitinases is most likely to take place through a substrate-assisted mechanism. Hen egg white lysozyme has the binding cleft represented by (-4)(-3)(-2)(-1)(+1)(+2). The binding cleft of family 19 chitinases, family 46 chitosanases, and family 23 lysozymes, however, is represented by (-3)(-2)(-1)(+1)(+2)(+3). Molecular dynamics calculation suggests that family 18 chitinases have the binding cleft, (-4)(-3)(-2)(-1)(+1)(+2). The functional diversity of the chitinolytic enzymes might be related to different physiological functions of the enzymes. The enzymes are now being applied to plant protection from fungal pathogens and insect pests. Structure of the targeted chitinous component was determined by a combination of enzyme digestion and solid state CP/MAS NMR spectroscopy, and have been taken into consideration for efficient application of the enzymes. Recent understanding of the catalytic and substrate binding mechanisms would be helpful as well for arrangement of a powerful strategy in such an application
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