Andrey V. Kajava

The leucine-rich repeat as a protein recognition motif.

Leucine-rich repeats (LRRs) are 20-29-residue sequence motifs present in a number of proteins with diverse functions. The primary function of these motifs appears to be to provide a versatile structural framework for the formation of protein-protein interactions. The past two years have seen an explosion of new structural information on proteins with LRRs. The new structures represent different LRR subfamilies and proteins with diverse functions, including GTPase-activating protein rna1p from the ribonuclease-inhibitor-like subfamily; spliceosomal protein U2A, Rab geranylgeranyltransferase, internalin B, dynein light chain 1 and nuclear export protein TAP from the SDS22-like subfamily; Skp2 from the cysteine-containing subfamily; and YopM from the bacterial subfamily. The new structural information has increased our understanding of the structural determinants of LRR proteins and our ability to model such proteins with unknown structures, and has shed new light on how these proteins participate in protein-protein interactions.

When protein folding is simplified to protein coiling: the continuum of solenoid protein structures

Solenoid proteins contain repeating structural units that form a continuous superhelix. This category of proteins conveys the least complicated relationship between a sequence and the corresponding three-dimensional structure. Although solenoid proteins are divided into different classes according to commonly used classification schemes, they share many structural and functional properties.

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500‐Å‐long secreted protein that is rich in β‐structure and contains two regions, R1 and R2, of tandem 19‐residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel β‐sheets; or (ii) a β‐helix in which the polypeptide chain is coiled to form three long parallel β‐sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional ‘covert’ copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the β‐helix model that the shaft domain – a 350u2003Å rod – should consist of a continuous run of these motifs, apart from loop inserts. The N‐terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin‐like structure. Drawing on crystal structures of known β‐helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins – all large secreted proteins of bacterial provenance – that have similar repeats and probably also similar structures.

Assessment of the ability to model proteins with leucine‐rich repeats in light of the latest structural information

The three‐dimensional structures of leucine‐rich repeat (LRR)‐containing proteins from five different families were previously predicted based on the crystal structure of the ribonuclease inhibitor, using an approach that combined homology‐based modeling, structure‐based sequence alignment of LRRs, and several rational assumptions. The structural models have been produced based on very limited sequence similarity, which, in general, cannot yield trustworthy predictions. Recently, the protein structures from three of these five families have been determined. In this report we estimate the quality of the modeling approach by comparing the models with the experimentally determined structures. The comparison suggests that the general architecture, curvature, “interior/exterior” orientations of side chains, and backbone conformation of the LRR structures can be predicted correctly. On the other hand, the analysis revealed that, in some cases, it is difficult to predict correctly the twist of the overall super‐helical structure. Taking into consideration the conclusions from these comparisons, we identified a new family of bacterial LRR proteins and present its structural model. The reliability of the LRR protein modeling suggests that it would be informative to apply similar modeling approaches to other classes of solenoid proteins.

What Curves α-Solenoids? EVIDENCE FOR AN α-HELICAL TOROID STRUCTURE OF Rpn1 AND Rpn2 PROTEINS OF THE 26 S PROTEASOME

The α-helical solenoid proteins adopt a variety of elongated curved structures. They have been examined to identify the interactions that determine their curvature. A sequence pattern characteristic for strongly curved α-helical solenoids has been constructed and was found to match protein sequences containing the proteasome/cyclosome repeats. Based on this, a structural model of the repeat-containing domains of the Rpn1/S2 and Rpn2/S1 proteins, which represent the largest subunits of the 26u2009S proteasome, has been proposed. The model has a novel architecture resembling an α-helical toroid. Molecular modeling shows that these toroids have a central pore that would allow passage of an unfolded protein substrate through it. This implies that the Rpn1 and Rpn2 toroids are aligned along the common axial pores of the ATPase hexamer and form an “antechamber” of the 26u2009S proteasome. The proposed quaternary structure agrees with the available experimental data. It is suggested that the function of this antechamber is assistance to the ATPases in the unfolding of protein substrates prior to proteolysis. An evolutionary link between the PC repeat-containing proteins and tetratricopeptide repeat proteins is proposed.

Redesign of a Four-Helix Bundle Protein by Phage Display Coupled with Proteolysis and Structural Characterization by NMR and X-ray Crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the proteins physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the proteins global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.