Elisabeth Sauer-Eriksson

Structural distribution of mutations associated with familial amyloidotic polyneuropathy in human transthyretin.

The human plasma protein transthyretin (TTR) is a highly stable soluble homotetrameric protein. Still, confor-mational changes in the wild type protein can lead to self-assembly into insoluble amyloid fibrils. In addition, 74 point mutations are known to enhance amyloid formation causing familial amyloidotic polyneuropathy (FAP). Alignment of TTR sequences from twenty different species shows that only six of these mutations occur as natural amino acids in other organisms. In this paper we analyse the distribution of FAP mutations within the three-dimensional structure of TTR. Contradictoty to what might be expected from protein stability studies, the mutations are not restricted to structurally rigid parts of the molecule, nor are they concentrated at the monomer interaction sites.

Structure of FocB--a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli.

In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form homomeric or heteromeric complexes and exert both positive and negative effects on the transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 Å resolution. FocB is an all α‐helical protein with a helix‐turn‐helix motif. Interestingly, conserved residues important for DNA‐binding are located not in the postulated recognition helix of the motif, but in the preceding helix. Results from protein–DNA‐binding studies suggest that FocB interacts with the minor groove of its cognate DNA target, which is indicative of a DNA interaction that is unusual for this motif. FocB crystallizes in the form of dimers. Packing interactions in the crystals give two plausible dimerization interfaces. Conserved residues, known to be important for protein oligomerization, are present at both interfaces, suggesting that both sites could play a role in a functional FocB protein.

Calcium binding by the PKD1 domain regulates interdomain flexibility in Vibrio cholerae metalloprotease PrtV

Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre‐pro‐protein that undergoes several N‐ and C‐terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755–838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca2+‐binding site located near linker regions between domains. Conformational changes in the Ca2+‐free and Ca2+‐bound forms suggest that Ca2+‐binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.

Crystallization and preliminary X-ray diffraction studies of the signal recognition particle receptor FtsY from Mycoplasma mycoides

The prokaryotic signal recognition particle (SRP) pathway comprises two proteins, Ffh and FtsY, homologous to the SRP54 and SRalpha proteins in the more complex eukaryotic system. All four proteins are part of a unique subfamily of GTPases. Four truncated versions of the 412 amino-acid FtsY receptor protein from Mycoplasma mycoides have been cloned, expressed in Escherichia coli and purified. Purified proteins from all constructs and the full-length FtsY protein were subjected to crystallization trials. Crystals were obtained for the construct which comprised residues 98-412 corresponding to the conserved NG-domain (residues 194-497 in E. coli). A native data set at 1.9 A resolution has been collected at 100 K using synchrotron radiation. The crystals belong to the space group P2(1)2(1)2, with unit-cell parameters a = 68.7, b = 101.1, c = 42.5 A and one molecule in the asymmetric unit.

Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli.

The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His(6)-tagged fusion protein was captured by Ni(2+)-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His(6) tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 A resolution was collected at 100 K using synchrotron radiation.

Assembly and Function of the Signal Recognition Particle from Archaea

The signal recognition particle (SRP) is a protein-RNA complex that associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. The universally conserved core of SRP consists of SRP RNA and the SRP54 protein, and plays the key role in signal-sequence recognition and binding to the SRP receptor. Critical for SRP function is communication between the two conserved SRP54 domains, the GTPase- and the M-domain, so that signal-sequence binding at the M domain directs receptor binding at the GTPase domain. The structural basis for signal-sequence binding by SRP and subsequent signaling is still poorly understood. By studying the structures of the SRP RNA in its free form as well as in complex with its different protein partners, we have made steady progress towards the elucidation of structural states of the SRP, using the archaeon Methanococcus jannaschii as model system. Together with other structures of SRP proteins and RNA-protein complexes, these structures provide new insights into the mechanisms of SRP-mediated protein targeting.

The multiple personalities of transthyretin

Engineering immunity against HIV-1 using designed antibody constructs Pamela J. Bjorkman, Michael A. Anaya, Shiyu Bai, Ron Diskin, Timothy J. Feliciano, Erin P. Flanagin, Rachel P. Galimidi, Han Gao, Priyanthi N. P. Gnanapragasam, Jennifer R. Keeffe, Terri Lee, Paola M. Marcovecchio, Maria D. Politzer, Anthony P. West, Jr., Yunji Wu, Division of Biology, California Institute of Technology, Pasadena, California (USA). E-mail: bjorkman@caltech.edu

Conformational change and assembly of proteins into amyloid fibrils

Self-assembly and deposition of proteins into amyloid fibrils and plaques have currently been linked to around 20 different human diseases. The best known examples of such disorders are Alzheimers disease and prion diseases. Amyloid contains extremely insoluble protein fibrils (50150 Å) that share similar morphological features but comprise many different proteins with no obvious sequence similarity. Amyloid formation and deposition are complex processes yet to be fully understood. Evidence from numerous in vitro studies shows that amyloid formation is a multistep process involving amyloidogenic intermediates. Structural and biophysical studies on amyloid-forming proteins are pursued with the aim to elucidate further information about the structural composition of amyloid and amyloid-forming intermediates. In this talk I will review some of our work with reference to three amyloidogenic proteins: amyloidβ-peptide, medin, and transthyretin.