Michael P. Williamson

The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

A common focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two‐hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to ones favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3‐dimensional analyses of antigen‐antibody interactions have revealed that epitopes are typically 4–7 residues long (1). Other examples include protein‐interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc‐large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14–3–3 proteins that typically recognize linear regions of 3–9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2–7). Among the primary structures of many ligands for protein–protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein‐interaction domains prefer ligand sequences that are proline‐rich. In addition, even though ligands for EH domains and 14–3–3 domains are not proline‐rich, they do include a single proline residue. This review highlights the analysis of those protein‐protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.—Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline‐rich motifs in signaling proteins with their cognate domains. FASEB J. 14, 231–241 (2000)

Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry.

A determination of the solution conformation of the proteinase inhibitor IIA from bull seminal plasma (BUSI IIA) is described. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain a list of 202 distance constraints between individually assigned hydrogen atoms of the polypeptide chain, to identify the positions of the three disulfide bridges, and to locate the single cis peptide bond. Supplementary geometric constraints were derived from the vicinal spin-spin couplings and the locations of certain hydrogen bonds, as determined by nuclear magnetic resonance (n.m.r.). Using a new distance geometry program (DISGEO) which is capable of computing all-atom structures for proteins the size of BUSI IIA, five conformers were computed from the NOE distance constraints alone, and another five were computed with the supplementary constraints included. Comparison of the different structures computed from the n.m.r. data among themselves and with the crystal structures of two homologous proteins shows that the global features of the conformation of BUSI IIA (i.e. the overall dimensions of the molecule and the threading of the polypeptide chain) were well-defined by the available n.m.r. data. In the Appendix, we describe a preliminary energy refinement of the structure, which showed that the constraints derived from the n.m.r. data are compatible with a low energy spatial structure.

Temperature dependence of 1H chemical shifts in proteins

Temperature coefficients have been measured by 2D NMR methods forthe amide and CαH proton chemical shifts in two globularproteins, bovine pancreatic trypsin inhibitor and hen egg-white lysozyme.The temperature-dependent changes in chemical shift are generally linear upto about 15° below the global denaturation temperature, and the derivedcoefficients span a range of roughly −16 to +2 ppb/K for amide protonsand −4 to +3 ppb/K for CαH. The temperaturecoefficients can be rationalized by the assumption that heating causesincreases in thermal motion in the protein. Precise calculations oftemperature coefficients derived from protein coordinates are not possible,since chemical shifts are sensitive to small changes in atomic coordinates.Amide temperature coefficients correlate well with the location of hydrogenbonds as determined by crystallography. It is concluded that a combined useof both temperature coefficients and exchange rates produces a far morereliable indicator of hydrogen bonding than either alone. If an amide protonexchanges slowly and has a temperature coefficient more positive than−4.5 ppb/K, it is hydrogen bonded, while if it exchanges rapidly andhas a temperature coefficient more negative than −4.5 ppb/K, it is nothydrogen bonded. The previously observed unreliability of temperaturecoefficients as measures of hydrogen bonding in peptides may arise fromlosses of peptide secondary structure on heating.

Polyphenols, astringency and proline-rich proteins

Recent, NMR and precipitation, studies of molecular recognition of proline-rich proteins and peptides by plant polyphenols are described and rationalized. The action of polysaccharides and caseins in the moderation of the astringent response, which is engendered by polyphenols present in foodstuffs and beverages, is described. The possible influence of plant cell wall glycoproteins on the process of lignification is discussed in the light of the observed affinity of phenolic substrates for prolyl residues in protein structures.

Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to β-cyclodextrin

BACKGROUND Carbohydrate-binding domains are usually small and physically separate from the catalytic domains of hydrolytic enzymes. Glucoamylase 1 (G1) from Aspergillus niger, an enzyme used widely in the food and brewing industries, contains a granular starch binding domain (SBD) which is separated from the catalytic domain by a semi-rigid linker. The aim of this study was to determine how the SBD binds to starch, and thereby more generally to throw light on the role of carbohydrate-binding domains in the hydrolysis of insoluble polysaccharides. RESULTS The solution structure of the SBD of A. niger G1 bound to beta-cyclodextrin (betaCD), a cyclic starch analogue, shows that the well-defined beta-sheet structure seen in the free SBD is maintained in the SBD-betaCD complex. The main differences between the free and bound states of the SBD are observed in loop regions, in or near the two starch-binding sites. The two binding sites, each of which binds one molecule of betaCD, are structurally different. Binding site 1 is small and accessible, and its structure changes very little upon ligand binding. Site 2 is longer and undergoes a significant structural change on binding. Part of this site comprises a flexible loop, which appears to allow the SBD to bind to starch strands in a range of orientations. CONCLUSIONS The two starch-binding sites of the SBD probably differ functionally as well as structurally; site 1 probably acts as the initial starch recognition site, whereas site 2 is involved in specific recognition of appropriate regions of starch. The two starch strands are bound at approximately 90 degrees to each other. This may be functionally important, as it may force starch strands apart thus increasing the hydrolyzable surface, or alternatively it may localize the enzyme to noncrystalline (more hydrolyzable) areas of starch. The region of the SBD where the linker to the catalytic domain is attached is flexible, allowing the catalytic site to access a large surface area of the starch granules.

The starch-binding domain from glucoamylase disrupts the structure of starch.

The full‐length glucoamylase from Aspergillus niger, G1, consists of an N‐terminal catalytic domain followed by a semi‐rigid linker (which together constitute the G2 form) and a C‐terminal starch‐binding domain (SBD). G1 and G2 both liberate glucose from insoluble corn starch, although G2 has a rate 80 times slower than G1. Following pre‐incubation of the starch with SBD, the activity of G1 is uniformly reduced with increasing concentrations of SBD because of competition for binding sites. However, increasing concentrations of SBD produce an initial increase in the catalytic rate of G2, followed by a decrease at higher SBD concentrations. The results show that SBD has two functions: it binds to the starch, but it also disrupts the surface, thereby enhancing the amylolytic rate.

The Structural Basis for the Ligand Specificity of Family 2 Carbohydrate-binding Modules

The interactions of proteins with polysaccharides play a key role in the microbial hydrolysis of cellulose and xylan, the most abundant organic molecules in the biosphere, and are thus pivotal to the recycling of photosynthetically fixed carbon. Enzymes that attack these recalcitrant polymers have a modular structure comprising catalytic modules and non-catalytic carbohydrate-binding modules (CBMs). The largest prokaryotic CBM family, CBM2, contains members that bind cellulose (CBM2a) and xylan (CBM2b), respectively. A possible explanation for the different ligand specificity of CBM2b is that one of the surface tryptophans involved in the protein-carbohydrate interaction is rotated by 90° compared with its position in CBM2a (thus matching the structure of the binding site to the helical secondary structure of xylan), which may be promoted by a single amino acid difference between the two families. Here we show that by mutation of this single residue (Arg-262→Gly), a CBM2b xylan-binding module completely loses its affinity for xylan and becomes a cellulose-binding module. The structural effect of the mutation has been revealed using NMR spectroscopy, which confirms that Trp-259 rotates 90° to lie flat against the protein surface. Except for this one residue, the mutation only results in minor changes to the structure. The mutated protein interacts with cellulose using the same residues that the wild-type CBM2b uses to interact with xylan, suggesting that the recognition is of the secondary structure of the polysaccharide rather than any specific recognition of the absence or presence of functional groups.

Tannin interactions with a full‐length human salivary proline‐rich protein display a stronger affinity than with single proline‐rich repeats

The protein IB5 has been purified from human parotid saliva. This protein contains several repeats of a short proline‐rich sequence. Dissociation constants have been measured at several discrete binding sites using 1H‐NMR for the hydrolysable tannins (polyphenols) ( , and and the condensed proanthocyanidin (−)‐epicatechin. The dissociation constants for trigalloyl glucose and pentagalloyl glucose were 15 × 10−5 and 1.7 × 10−5 M, respectively, which are 115 and 1660 times stronger than those previously measured under the same conditions for a single repeat of a mouse salivary proline‐rich protein. The increase in affinity is ascribed to intramolecular secondary interactions, which are strengthened by the rigidity of the interacting molecules.

The Campylobacter jejuni PEB1a adhesin is an aspartate/glutamate-binding protein of an ABC transporter essential for microaerobic growth on dicarboxylic amino acids

The PEB1a protein of the gastrointestinal pathogen Campylobacter jejuni mediates interactions with epithelial cells and is an important factor in host colonization. Cell fractionation and immunoblotting showed that PEB1a is most abundant in the periplasm of C. jejuni, and is detectable in the culture supernatant but not in the inner or outer membrane. The protein is homologous with periplasmic‐binding proteins associated with ABC transporters and we show by fluorescence spectroscopy that purified recombinant PEB1a binds l‐aspartate and l‐glutamate with sub µM Kd values. Binding of l‐14C‐aspartate or l‐14C‐glutamate was strongly out‐competed by excess unlabelled aspartate or glutamate but only poorly by asparagine and glutamine. A mutant in the Cj0921c gene, encoding PEB1a, was completely unable to transport 5 µM l‐14C‐glutamate and showed a large reduction (∼20‐fold) in the rate of l‐14C‐aspartate transport compared with the wild type. Although microaerobic growth of this mutant was little affected in complex media, growth on aspartate or glutamate in defined media was completely prevented, whereas growth with serine was similar to wild type. 1H‐NMR analysis of the culture supernatants of the Cj0921c mutant showed some utilization of aspartate but not glutamate, consistent with the transport data. It is concluded that in addition to the established role of PEB1a as an adhesin, the PEB1 transport system plays a key role in the utilization of aspartate and glutamate, which may be important in vivo carbon sources for this pathogen.

The relationship between amide proton chemical shifts and secondary structure in proteins

SummaryThe parameters for HN chemical shift calculations of proteins have been determined using data from high-resolution crystal structures of 15 proteins. Employing these chemical shift calculations for HN protons, the observed secondary structure chemical shift trends of HN protons, i.e., upfield shifts on helix formation and downfield shifts on β-sheet formation, are discussed. Our calculations suggest that the main reason for the difference in NH chemical shifts in helices and sheets is not an effect from the directly hydrogen-bonded carbonyl, which gives rise to downfield shifts in both cases, but arises from an additional upfield shift predicted in helices and originating in residues i-2 and i-3. The calculations also explain the well-known relationship between amide proton shifts and hydrogen-bond lengths. In addition, the HN chemical shifts of the distorted amphipathic helices of the GCN4 leucine zipper are calculated and used to characterise the solution structure of the helices. By comparing the calculated and experimental shifts, it is shown that in general the agreement is good between residues 15 and 28. The most interesting observation is that in the N-terminal half of the zipper, although both calculated and experimental shifts show clear periodicity, they are no longer in phase. This suggests that for the N-terminal half, in the true average solution structure the period of the helix coil is longer by roughly one residue compared to the NMR structures.