Johan Kemmink

The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules

BACKGROUNDnProtein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic reticulum, catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. Dissection of this protein into its individual domains has confirmed the presence of the a and a domains, which are homologous to thioredoxin, having related structures and activities. The a and a domains both contain a -Cys-Gly-His-Cys- active-site sequence motif. The remainder of the molecule consists primarily of two further domains, designated b and b which are thought to be sequence repeats on the basis of a limited sequence similarity. The functions of the b and b domains are unknown and, until now, the structure of neither domain was known.nnnRESULTSnHeteronuclear nuclear magnetic resonance (NMR) methods have been used to determine the global fold of the PDI b domain. The protein has an alpha/beta fold with the order of the elements of secondary structure being beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-beta5+ ++-alpha4. The strands are all in a parallel arrangement with respect to each other, except for beta4 which is antiparallel. The arrangement of the secondary structure elements of the b domain is identical to that found in the a domain of PDI and in the ubiquitous redox protein thioredoxin; the three-dimensional folding topology of the b domain is also very similar to that of these proteins.nnnCONCLUSIONSnOur determination of the global fold of the b domain of PDI by NMR reveals that, like the a domain, the b domain contains the thioredoxin motif, even though the b domain has no significant amino-acid sequence similarities to any members of the thioredoxin family. This observation, together with indications that the b domain adopts a similar fold, suggests that PDI consists of active and inactive thioredoxin modules. These modules may have been adapted during evolution to provide PDI with its complete spectrum of enzymatic activities.

Letter to the Editor: Sequence-specific assignment and determination of the secondary structure of the 163-residue M. tuberculosis and M. bovis antigenic protein mpb70

Tuberculosis is one of the most significant bacterial diseases of humans, with about one third of the world’s population infected (Kochi, 1994). The complete sequence of the M. tuberculosis genome has recently been determined and codes for 3924 proteins (Cole et al., 1998), and the M. bovis genome will shortly be available. However, we still have very little information about which proteins are essential for pathogenesis and even less knowledge of their structures, functions and mechanisms of action. The mature M. bovis protein mpb70 (163 residues) is an immunodominant antigen, which is secreted from M. bovis cells following cleavage of a 30-residue signal peptide (Hewinson and Russell, 1993). The M. tuberculosis homologue of mpb70 is mpt70 and the sequences of the two proteins are identical (Cole et al., 1998). Virulent M. bovis expresses high levels of mpb70 and although the expression of mpt70 is low in M. tuberculosis cells grown in vitro, both mycobacteria stimulate a strong immune response to mpb70/mpt70 on infection (Hewinson and Russell, 1993; Hewinson et al., 1996). In addition, treatment of M. tuberculosis infected mice with a DNA vaccine encoding mpb70 has a pronounced therapeutic action (Lowrie et al., 1999). Together, this suggests that mpb70 may be required for the survival of M. tuberculosis and M. bovis in vivo. ∗To whom correspondence should be addressed. E-mail: M.D.Carr@ukc.ac.uk (M.D.C.); R.A.Williamson@ukc.ac.uk (R.A.W.) To date, the function of mpb70 and its role in tuberculosis pathogenesis remains unknown. In this letter we report the determination of sequence-specific backbone resonance assignments for mpb70 and identification of the secondary structure of the protein. The continuation of this work will lead to the determination of a high resolution structure for mpb70, which will assist in determining its function and role in tuberculosis pathogenesis.

Preferred Local Conformations of Peptides comprising the Entire Sequence of Bovine Pancreatic Trypsin Inhibitor (BPTI) and their Roles in Protein Folding

Unfolded single-domain globular proteins generally fold spontaneously into their native three-dimensional structures. As a consequence, the three-dimensional fold of a polypeptide chain must be determined by its primary structure, the amino-acid sequence. Considering the time scale of the folding process, which is of the order of 10−1–10+2 s for small proteins, folding is unlikely to proceed by a random sampling of all possible conformations. The folding pathway of a protein is determined by (1) the nature of the initial unfolded protein under refolding conditions and (2) the kinetic intermediate states through which the protein molecules pass transiently when folding.