Mark Bycroft

The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold.

The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins. The structure of the S1 RNA-binding domain from the E. coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel. Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site. The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein. Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins.

The macro domain is an ADP-ribose binding module.

The ADP‐ribosylation of proteins is an important post‐translational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long‐term memory formation. Yet no protein modules are known that specifically recognize the ADP‐ribose nucleotide. We provide biochemical and structural evidence that macro domains are high‐affinity ADP‐ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP‐ribose. In addition, some macro domain proteins also recognize poly‐ADP‐ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP‐ribose.

Strength and co-operativity of contributions of surface salt bridges to protein stability.

Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.

RNA recognition by a Staufen double-stranded RNA-binding domain

The double‐stranded RNA‐binding domain (dsRBD) is a common RNA‐binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA‐binding activity of dsRBD3 is required in vivo for Staufen‐dependent localization of bicoid and oskar mRNAs. Using high‐resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A‐form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single‐stranded loop that caps the RNA helix. Interactions between helix α1 and single‐stranded RNA may be important determinants of the specificity of dsRBD proteins.

Molecular Mechanism of the Interaction between MDM2 and p53

We have investigated the kinetic and thermodynamic basis of the p53-MDM2 interaction using a set of peptides based on residues 15-29 of p53. Wild-type p53 peptide bound MDM2 with a dissociation constant of 580nM. Phosphorylation of S15 and S20 did not affect binding, but T18 phosphorylation weakened binding tenfold, indicating that phosphorylation of only T18 is responsible for abrogating p53-MDM2 binding. Truncation to residues 17-26 increased affinity 13-fold, but further truncation to 19-26 abolished binding. NMR studies of the binding of the p53-derived peptides revealed global conformational changes of the overall structure of MDM2, stretching far beyond the binding cleft, indicating significant changes in the domain dynamics of MDM2 upon ligand binding.

Aromatic-aromatic interactions and protein stability: Investigation by double-mutant cycles

The side-chains of phenylalanine and tyrosine residues in proteins are frequently found to be involved in pairwise interactions. These occur both within repeating elements of secondary structure and in tertiary and quaternary interactions. It has been suggested that they are important in protein folding and stability, and non-bonded potential energy calculations indicate that a typical aromatic-aromatic interaction has an energy of between -1 and -2 kcal/mol and contributes between -0.6 and -1.3 kcal/mol to protein stability. There is such an aromatic pair on the solvent-exposed face of the first alpha-helix of barnase, the small ribonuclease from Bacillus amyloliquefaciens. The edge of the aromatic ring of Tyr17 interacts with the face of that of Tyr13. The two residues have been mutated both singly and pairwise to alanine, and their free energies of unfolding determined by denaturation with urea. Application of the double-mutant cycle analysis gives an interaction energy of -1.3 kcal/mol for the aromatic pair in the folded protein relative to solvation by water in the unfolded protein. This value is similar to that calculated from the change in surface-accessible area between the rings on the formation of the pair. Analysis of a further double-mutant cycle in which the Tyr residues are mutated to Phe indicates that the aromatic-aromatic interactions of Tyr/Tyr and Phe/Phe make identical contributions to protein stability. However, Tyr is preferred to Phe by 0.3(+/- 0.04) kcal/mol at the solvent-exposed face of the alpha-helix.

Crystal Structure of a Calcium-Phospholipid Binding Domain from Cytosolic Phospholipase A2*

Cytosolic phospholipase A2 (cPLA2) is a calcium-sensitive 85-kDa enzyme that hydrolyzes arachidonic acid-containing membrane phospholipids to initiate the biosynthesis of eicosanoids and platelet-activating factor, potent inflammatory mediators. The calcium-dependent activation of the enzyme is mediated by an N-terminal C2 domain, which is responsible for calcium-dependent translocation of the enzyme to membranes and that enables the intact enzyme to hydrolyze membrane-resident substrates. The 2.4-Å x-ray crystal structure of this C2 domain was solved by multiple isomorphous replacement and reveals a β-sandwich with the same topology as the C2 domain from phosphoinositide-specific phospholipase Cδ1. Two clusters of exposed hydrophobic residues surround two adjacent calcium binding sites. This region, along with an adjoining strip of basic residues, appear to constitute the membrane binding motif. The structure provides a striking insight into the relative importance of hydrophobic and electrostatic components of membrane binding for cPLA2. Although hydrophobic interactions predominate for cPLA2, for other C2 domains such as in “conventional” protein kinase C and synaptotagmins, electrostatic forces prevail.

NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.

The double‐stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double‐stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA‐dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha‐beta‐beta‐beta‐alpha structure in which the two alpha‐helices lie on one face of a three‐stranded anti‐parallel beta‐sheet. This structure is very similar to that of the N‐terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.

The structure of a PKD domain from polycystin‐1: implications for polycystic kidney disease

Most cases of autosomal dominant polycystic kidney disease (ADPKD) are the result of mutations in the PKD1 gene. The PKD1 gene codes for a large cell‐surface glycoprotein, polycystin‐1, of unknown function, which, based on its predicted domain structure, may be involved in protein–protein and protein–carbohydrate interactions. Approximately 30% of polycystin‐1 consists of 16 copies of a novel protein module called the PKD domain. Here we show that this domain has a β‐sandwich fold. Although this fold is common to a number of cell‐surface modules, the PKD domain represents a distinct protein family. The tenth PKD domain of human and Fugu polycystin‐1 show extensive conservation of surface residues suggesting that this region could be a ligand‐binding site. This structure will allow the likely effects of missense mutations in a large part of the PKD1 gene to be determined.

Surface electrostatic interactions contribute little to stability of barnase

Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second alpha-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 0.2 kcal/mol to stability of the protein. All other interactions between exposed charged side-chains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein.