Current Protein and Peptide Science - Volume 4, Issue 5, 2003

Regulation of proteolytic enzyme activity is an essential requirement for cells and tissues because proteolysis at the wrong time and location may be lethal. Two principal mechanisms to control the activity of proteases have been developed during evolution. The first is the co-evolution of endogenous inhibitors, typically occurring in cellular compartments separated from those containing active enzymes. The second is the fact that proteases are synthesized as inactive or less active precursor molecules. They are activated, in some cases, upon an appropriate signal like acidification, Ca++ -binding or, in other cases, by limited intra- or intermolecular proteolysis cleaving off an inhibitory peptide. These regulatory proenzyme regions have attracted much attention during the last decade, since it became obvious that they harbour much more information than just triggering activation. In this review we summarize experimental data concerning three functions of propeptides of clan CA family C1 cysteine peptidases (papain family), namely the selectivity of their inhibitory potency, the participation in correct intracellular targeting and assistance in folding of the mature enzyme. Cysteine peptidases of the CA-C1 family include members from the plant kingdom like papain as well as from the animal kingdom like the lysosomal cathepsins L and B. As it will be shown, the functions are determined by certain structural motifs conserved over millions of years after the evolutionary trails have diverged. The function of propeptides of two other important classes of cysteine peptidases - the calpains, clan CA family C4, and the caspases, clan CD family C 14 - are not considered in this review.

Type II restriction endonucleases (ENases) cleave DNA with remarkable sequence specificity. Their discovery in 1970 and studies on molecular genetics and biochemistry carried out over the past four decades laid foundations for recombinant DNA techniques. Today, restriction enzymes are indispensable tools in molecular biology and molecular medicine and a paradigm for proteins that specifically interact with DNA as well as a challenging target for protein engineering. The sequence-structure-function relationships for these proteins are therefore of central interest in biotechnology. However, among numerous ENase sequences, only a few exhibit statistically significant similarity in pairwise comparisons, which was initially interpreted as evidence for the lack of common origin. Nevertheless, X-ray crystallographic studies of seemingly dissimilar type II ENases demonstrated that they share a common structural core and metal-binding / catalytic site, arguing for extreme divergence rather than independent evolution. A similar nuclease domain has been also identified in various enzymes implicated in DNA repair and recombination. Ironically, following the series of crystallographic studies suggesting homology of all type II ENases, bioinformatic studies provided evidence that some restriction enzymes are in fact diverged members of unrelated nuclease superfamilies: Nuc, HNH and GIY-YIG. Hence, the restriction enzymes as a whole, represent a group of functionally similar proteins, which evolved on multiple occasions and subsequently diverged into the “midnight zone” of homology, where common origins within particular groups can be inferred only from structure-guided comparisons. The structure-guided approaches used for this purpose include: identification of functionally important residues using superposition of atomic coordinates, alignment of sequence profiles enhanced by secondary structures, fold recognition, and homology modeling. This review covers recent results of comparative analyses of restriction enzymes from the four currently known superfamilies of nucleases with distinct folds, using crystallographic and bioinformatic methods, with the emphasis on theoretical predictions and their experimental validation by site-directed mutagenesis and biochemical analyses of the mutants.

Physiological processes are regulated to a large extent by physical and chemical interactions between polypeptides. Although many small molecules have been discovered that can modulate such interactions and may be useful as drugs, the design of these agents purely from the knowledge of the details of a given protein-protein interaction, or through screening, remains difficult. Therefore, the peptidomimetic process, which aims at using peptides derived from either polypeptide binding partner directly, or after modification to improve affinity and physicochemical properties, continues to be attractive. The vast majority of naturally occurring polypeptides are composed of L-amino acids. Because natural proteins need to be metabolised, L-amino acid polypeptides are very prone to proteolytic degradation, a property that severely limits their therapeutic application. The proteolytic machinery is not well equipped to deal with D-amino acid polypeptides, however, and it is this finding above all else that has spurned research into stereochemical and directional manipulation of peptide chains. The expectation has been that systematic inversion of the stereochemistry at the peptide backbone α-carbon atoms, if accompanied by chain reversal, should yield proteolytically stable retro-inverso peptide isomers, whose side chain topology, in the extended conformation, corresponds closely to that of a native sequence, and whose biological activity emulates that of a parent polypeptide. The actual structural implications of modifying amino acid stereochemistry and peptide bond direction are reviewed critically here and the reasons for the lack of general success with this strategy are discussed. The application of polypeptides is particularly pertinent to synthetic vaccine design. Interestingly, the retro-inverso strategy has been more successful for immunological applications than elsewhere; recent finding are collated in this review. Partial rather than global retro-inversion holds much promise since the loss of crucial backbone hydrogen-bonding through peptide bond reversal can be avoided, while still permitting stabilisation of selected hydrolysis-prone peptide bonds. Generically applicable synthetic methods for such partially modified retro-inverso peptides are not as yet available; progress towards this goal is also summarised.

Amphiphilic α-helices play a fundamental role in protein - membrane association and show a segregation of polar and apolar amino acid residues. Based on correlations between amphiphilic properties and biological function, a number of theoretical approaches have been developed, which quantify α-helix amphiphilicity and then attempt to assign function. The most commonly used measure of amphiphilicity is the hydrophobic moment, < μH >, which, when used in conjunction with an α-helix's mean hydrophobicity, < H >, has been used to classify membrane interactive amphiphilic α- helices as either surface active or transmembrane. Here, the predictive efficacy of plot methodology is reviewed by examining published data, which compare the function of known membrane interactive amphiphilic α-helices to that assigned by this methodology. The results of this review are discussed in relation to the reliability of < μH > as a quantifier of α-helical amphiphilicity, and the ability of < μH > and < H > to describe α-helical structure / function relationships. It is concluded that hydrophobic moment plot methodology is not a generally reliable predictor of α-helical structure / function relationships. It appears that the inefficacy of plot methodology is primarily due to the inability of the plot diagram to accommodate the heterogeneity of the α-helical classes it attempts to define. However, the predictive efficacy of the methodology appears to be improved if other α-helical parameters are also considered when assigning α-helical function. It is suggested that the conventional methodology should be seen only as an indicator for the assignation of structure / function relationships, providing a guide to future experimental investigations.

CD2 (cluster of differentiation 2) is a cell adhesion molecule expressed on T cells and is recognized as a target for CD48 (rats) and CD58 (humans). Tremendous progress has been achieved in understanding the function of CD2, the mechanism of molecular recognition and protein folding, thus, leading towards the use of this protein as a scaffold for protein design. CD2 has been shown to set quantitative thresholds in T cell activation both in vivo and in vitro. Further, intracellular CD2 signaling pathways and networks are being discovered by the identification of several cytosolic tail binding proteins. In addition, a new method for directly measuring heterophilic adhesion has been developed. The functional “hot spot” for the adhesion surface of CD2 and CD58 has been dissected. Detailed NMR studies reveal that rat CD2 weakly self-associates to form a homodimeric structure in solution. Dynamic interaction of CD2 with the GYF and SH3 domains has been investigated. CD2 has been shown to form fibrils in the presence of 2,2,2-trifluoroethanol (TFE) and at low pH. Furthermore, kinetic studies have been completed to monitor the effect of surface hydrophobic residues and intramolecular bridges on the folding pathways of CD2. Our lab has de novo designed single calcium-binding sites into domain 1 of rat CD2 (CD2-D1) with strong metal selectivity. In addition, the EF-hand motifs have been grafted into CD2 to understand the site-specific calcium-binding affinity of calmodulin and calcium-dependent cell adhesion.

The recent breakthroughs in genomics and proteomics and improvements of optical methods have made it possible to obtain localized, real-time information on intracellular proteins dynamics, through dynamic three-dimensional (3D) maps of the living cell with nanometric resolution of individual molecules. On one side, brighter variants of the Green Fluorescence Protein (GFP) have been engineered that have different excitation and / or emission spectra that better match available light sources. Like their parent molecule, these variants retain their fluorescence when fused to heterologous proteins on the N- and C- terminals, and this binding generally does not affect the functionality of the tagged protein leading the way to their use as an intracellular reporter. On the other side, optical methods have been improved to allow reaching the level of single-molecule detection inside living cells. Nevertheless some limitations exist for the use of GFP variants for probing 3D conformational changes of proteins. First, these variants are fused to the N and / or C terminals of the studied protein, which are generally not the best location to detect conformational changes resulting from the binding to other proteins or enzyme substrates. Then their own relatively large size makes them unusable for tagging small proteins. These limitations suggest that new tagging processes, permitting the location of the right fluorescent markers at the right places, must be found to built up inter- and / or intra-molecular rulers allowing one to monitor conformational changes resulting from intracellular protein-protein, protein-membrane, and enzyme-substrate binding. These specific locations can be obtained from in vitro studies of 3D conformational changes that occur during protein docking.