Structural
Biology of the Cell Adhesion Protein CD2: Alternatively Folded States and
Structure-function Relation Pp.1-17
Protein-X,
Pancreatic Stone-, Pancreatic Thread-, reg-Protein, P19, Lithostathine, and Now
What ?
Characterization,
Structural Analysis and Putative Function(s) of the Major Non-Enzymatic Protein
of Pancreatic Secretions Pp.19-42
M. De Reggi and B. Gharib
Structure
and Function of Complement Activating Enzyme Complexes: C1 and MBL-MASPs Pp.43-59
P. Gal and G. Ambrus
Protein
Thiol Modification of Glyceraldehyde-3-phosphate Dehydrogenase and Caspase-3 by
Nitric Oxide Pp.61-72
Bernhard Brüne and Susanne Mohr
Concepts
and Misconcepts in the Analysis of Simple Kinetics of Protein Folding Pp.73-98
Alexander P. Demchenko
[Back to top] Structural Biology of the Cell Adhesion
Protein CD2: Alternatively Folded States and Structure function Relation
Cluster of differentiation 2 (CD2) is a cell surface
glycoprotein expressed on most human T cells and natural killer (NK) cells and
plays an important role in mediating cell adhesion in both T-lymphocytes and in
signal transduction. The understanding of the biochemical basis of molecular
recognition by the cell adhesion molecule CD2 has been advanced greatly through
the determination of structures and the dynamic properties of the complexes and
their individual components and through site-directed
mutagenesis. A number of general principles can be derived from the structural
and functional studies of the extracellular domains of CD2 and CD58 and their
complex. Significant electrostatic interactions within the protein-protein interfaces
contribute directly to the formation of macromolecular complexes of CD2 and
CD58. Also, residues located on the protein-protein interface demonstrate a
certain degree of conformational change upon the formation of a complex.
Structural analysis of CD2 has revealed that this adhesion molecule exhibits
strong conformational flexibility with a partial non-native helical
conformation at high temperatures and in the presence of an organic solvent. In
addition, it can be converted into a domain swapped dimer, or trimer and
tetramer through hinge deletion. Thus, the conformational status of the
adhesive proteins contributes
to the regulation of cell adhesion and the folding of CD2.
[Back to top] Protein-X, Pancreatic
Stone-, Pancreatic Thread-, reg-Protein, P19, Lithostathine, and Now What ?
Characterization,
Structural Analysis and Putative Function(s) of the Major Non-Enzymatic Protein
of Pancreatic Secretions
M. De Reggi and B. Gharib
Reg protein was first found in pancreatic stones. It was
named Pancreatic Stone Protein and later renamed lithostathine, as it was
assumed to prevent stone formation. The 144 amino acid protein is
O-glycosylated on Thr-5. The glycan chain is variable in length and in charge.
Lithostathine 3-D organization is of the C-lectin type, even though it is
unlikely to have any functional calcium-binding site. The Arg11-Ile12 bond is
readily cleaved by trypsin; the resulting C-terminal polypeptide precipitates
at physiological pH and tends to form fibrils. The protein was more recently
found in the regenerating endocrine
pancreas and it was named Reg (for regenerating) protein. Numerous proteins
related to Reg have been identified successively in several mammalian species.
They constitute the Reg superfamily. Reg genes show the same organization and
are located in the same chromosome region. These genes are therefore likely to
derive from a common ancestor gene by duplication. In the course of evolution,
they may have diverged in tissue-related expression and function. In the
endocrine pancreas, Reg protein stimulates islet beta-cell growth and reduces
experimental diabetes via the activation of a high affinity receptor. The role
of the protein produced by the exocrine pancreas, however, is controversial.
Not only is Reg/lithostathine unlikely to be a physiologically relevant
pancreatic stone inhibitor, but it may contribute to stone formation. We
suggest that it might help prevent the harmful activation of protease precursors
in the pancreatic juice. The protein provides a useful model for examining the
conformational changes associated with globular to fibril transformation.
[Back to top] Structure and
Function of Complement Activating Enzyme Complexes: C1 and MBL-MASPs
P. Gal and G. Ambrus
The complement system is a major effector arm of the
immune defense contributing to the destruction of invading pathogens. There are
three possible routes of complement cascade activation: the classical, the
alternative and the lectin pathways. The
activation of the classical and lectin pathways is initiated by
supramolecular complexes, which resemble each other. Each complex has a
recognition subunit (C1q in the classical and mannose-binding lectin (MBL) in the lectin pathway), which
associates with serine protease zymogens (C1q with C1r and C1s, and MBL with
MBL-associated serine proteases: MASP-1, MASP-2) to form the C1 and MBL-MASPs
complexes, respectively. As the recognition subunits bind to activator
structures, subsequent activation of the serine protease zymogens occurs. The
precise structure of the complexes and the exact mechanism of their activation
have not been solved, yet.
In this review
we summarize the recent advances about the structure and function of the
individual subcomponents of both complexes achieved by genetic engineering,
molecular modeling, physico-chemical and functional studies. Special emphasis
will be laid on the serine proteases: the role of the individual domains in the
assembly of the C1s-C1r-C1r-C1s tetramer and in the control of the protease
activity will be discussed. We will then focus on recent functional models of
the supramolecular complexes. The question of how a non-enzymatic signal (the
binding of C1q or MBL to activators) can be converted into enzymatic events
(activation of serine protease zymogens) will be addressed. The similarities
and differences betweenC1 and MBL-MASPs will also be discussed.
[Back to top] Protein Thiol Modification of
Glyceraldehyde-3-phosphate Dehydrogenase and Caspase-3 by Nitric Oxide
Bernhard Brüne and Susanne Mohr
The regulation of enzyme activity function is a major
factor in the cellular response to a changing environment. One mechanism of
enzyme activity regulation includes post-translational protein thiol
modification by nitric oxide (NO) or its redox species. Major routs used by NO
to modify cysteine residues of proteins include S-nitrosation, oxidation, mixed
disulfide formation with glutathione, and the covalent attachment of nucleotide
cofactors, i.e NAD+/NADH. Critical thiol centers serve as
recognition sites for NO, thus channeling the NO signal through
post-translational modifications and oxidation into cellular functions. Here,
we summarize current knowledge on active site thiol modification of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and caspase-3 by nitric oxide.
Although very different in their cellular function, both enzymes contain highly
reactive cysteines which represent sensitive targets for NO. Our studies are
supportive of a potential role of S-nitrosation and mixed disulfide formation as a general signaling mechanism that
allows sensing of nitrosative stress. At the same time, modification of GAPDH
and caspase-3 by NO show the diversity of mechanisms (S-nitrosation versus
oxidations) that we are confronted with as a result of NO delivery, especially
comparing in vitro studies with cellular systems. In the future it will be
challenging to dissect how nitrosative and oxidative signaling mechanisms
overlap and how intracellular communication systems allow their activation in a
selective way.
[Back to top] Concepts and Misconcepts in the Analysis of
Simple Kinetics of Protein Folding
Alexander P. Demchenko
Unusually simple two-state kinetics characterizes the
folding of a number of small proteins possessing a variety secondary
structures. This limits dramatically the number of experimentally resolvable
parameters that may characterize this process and also suggests the possibility to describe it based on simple theories
borrowed from the field of ordinary chemical reactions. An attempt is made to
critically evaluate the basic concepts, which are in the background of this
approach. We demonstrate their limitations, which may cast doubt on the
interpretation of experimental data. It is shown also that, in contrast to
provisions of transition state theory, the simple kinetics of protein folding
does not correlate with folded state stability or with the size of the folding
unit. Moreover, the folding kinetics exhibits anomalous dependence on
temperature and pressure and surprisingly strong dependence on solvent
viscosity. The possible role in folding of fluctuations, relaxations and gradient
dynamics is discussed. Being overlooked or underestimated, these mechanisms may determine the rate and specificity
of the process.