Robert Janowski

Bacterioferritin from Mycobacterium smegmatis contains zinc in its di-nuclear site.

Bacterioferritins, also known as cytochrome b 1, are oligomeric iron‐storage proteins consisting of 24 identical amino acid chains, which form spherical particles consisting of 24 subunits and exhibiting 432 point‐group symmetry. They contain one haem b molecule at the interface between two subunits and a di‐nuclear metal binding center. The X‐ray structure of bacterioferritin from Mycobacterium smegmatis (Ms‐Bfr) was determined to a resolution of 2.7 Å in the monoclinic space group C2. The asymmetric unit of the crystals contains 12 protein molecules: five dimers and two half‐dimers located along the crystallographic twofold axis. Unexpectedly, the di‐nuclear metal binding center contains zinc ions instead of the typically observed iron ions in other bacterioferritins.

Structure and non-structure of centrosomal proteins

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php.

The structure of dihydrodipicolinate reductase (DapB) from Mycobacterium tuberculosis in three crystal forms.

Dihydrodipicolinate reductase (DHDPR, DapB) is an enzyme that belongs to the L-lysine biosynthetic pathway. DHDPR reduces the alpha,beta-unsaturated cyclic imine 2,3-dihydrodipicolinic acid to yield the compound 2,3,4,5-tetrahydrodipicolinic acid in a pyridine nucleotide-dependent reaction. The substrate of this reaction is the unstable product of the preceding enzyme dihydrodipicolinate synthase (DHDPS, DapA). Here, the structure of apo-DHDPR from Mycobacterium tuberculosis is reported in two orthorhombic crystal forms, as well as the structure of DHDPR from M. tuberculosis in complex with NADH in a monoclinic crystal form. A comparison of the results with previously solved structures of this enzyme shows that DHDPR undergoes a major conformational change upon binding of its cofactor. This conformational change can be interpreted as one of the low-frequency normal modes of the structure.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapB (Rv2773c) from Mycobacterium tuberculosis

Dihydrodipicolinate reductase from Mycobacterium tuberculosis (DapB, DHDPR, Rv2773c) has been cloned and heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in three different crystal forms. Preliminary diffraction data analysis suggests the presence of two tetramers in the asymmetric unit of one crystal form and half a tetramer in the other two crystal forms.

Structural Basis for Antiviral Inhibition of the Main Protease, 3C, from Human Enterovirus 93

ABSTRACT Members of the Enterovirus genus of the Picornaviridae family are abundant, with common human pathogens that belong to the rhinovirus (HRV) and enterovirus (EV) species, including diverse echo-, coxsackie- and polioviruses. They cause a wide spectrum of clinical manifestations ranging from asymptomatic to severe diseases with neurological and/or cardiac manifestations. Pandemic outbreaks of EVs may be accompanied by meningitis and/or paralysis and can be fatal. However, no effective prophylaxis or antiviral treatment against most EVs is available. The EV RNA genome directs the synthesis of a single polyprotein that is autocatalytically processed into mature proteins at Gln↓Gly cleavage sites by the 3C protease (3Cpro), which has narrow, conserved substrate specificity. These cleavages are essential for virus replication, making 3Cpro an excellent target for antivirus drug development. In this study, we report the first determination of the crystal structure of 3Cpro from an enterovirus B, EV-93, a recently identified pathogen, alone and in complex with the anti-HRV molecules compound 1 (AG7404) and rupintrivir (AG7088) at resolutions of 1.9, 1.3, and 1.5 Å, respectively. The EV-93 3Cpro adopts a chymotrypsin-like fold with a canonically configured oxyanion hole and a substrate binding pocket similar to that of rhino-, coxsackie- and poliovirus 3C proteases. We show that compound 1 and rupintrivir are both active against EV-93 in infected cells and inhibit the proteolytic activity of EV-93 3Cpro in vitro. These results provide a framework for further structure-guided optimization of the tested compounds to produce antiviral drugs against a broad range of EV species.

The structure of the complex between α-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism.

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well‐characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in &agr;‐tubulin–&bgr;‐tubulin dissociation. We used electron microscopy and image processing to determine the three‐dimensional structure of the human TBCE, TBCB and &agr;‐tubulin (&agr;EB) complex, which is formed upon &agr;‐tubulin–&bgr;‐tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X‐ray crystallography, allowed description of the molecular architecture of the &agr;EB complex. We found that heterodimer dissociation is an energy‐independent process that takes place through a disruption of the &agr;‐tubulin–&bgr;‐tubulin interface that is caused by a steric interaction between &bgr;‐tubulin and the TBCE cytoskeleton‐associated protein glycine‐rich (CAP‐Gly) and leucine‐rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin‐like (UBL) domains in the &agr;EB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating &agr;‐tubulin degradation.

The structure of the complex between a-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well‐characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in &agr;‐tubulin–&bgr;‐tubulin dissociation. We used electron microscopy and image processing to determine the three‐dimensional structure of the human TBCE, TBCB and &agr;‐tubulin (&agr;EB) complex, which is formed upon &agr;‐tubulin–&bgr;‐tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X‐ray crystallography, allowed description of the molecular architecture of the &agr;EB complex. We found that heterodimer dissociation is an energy‐independent process that takes place through a disruption of the &agr;‐tubulin–&bgr;‐tubulin interface that is caused by a steric interaction between &bgr;‐tubulin and the TBCE cytoskeleton‐associated protein glycine‐rich (CAP‐Gly) and leucine‐rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin‐like (UBL) domains in the &agr;EB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating &agr;‐tubulin degradation.

Reductive methylation to improve crystallization of the putative oxidoreductase Rv0765c from Mycobacterium tuberculosis

Rv0765c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and crystallized. The native protein crystallized in a hexagonal crystal form which diffracted to 7 A resolution. In an attempt to improve the quality of the Rv0765c crystals, the protein was modified by reductive methylation using dimethylaminoborane and formaldehyde. The modified protein crystallized under different conditions in a tetragonal crystal form, from which diffraction data could be collected to a resolution of 3.2 A. In both crystal forms of Rv0765c, the asymmetric unit contained two copies of the protein molecule.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv2827c from Mycobacterium tuberculosis.

The hypothetical protein Rv2827c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and then crystallized. Preliminary X-ray diffraction data analysis suggests the presence of two translationally related molecules in the asymmetric unit of the orthorhombic crystals.

The structure of the TBCE/TBCB chaperones and a-tubulin complex shows a tubulin dimer dissociation mechanism

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well‐characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in &agr;‐tubulin–&bgr;‐tubulin dissociation. We used electron microscopy and image processing to determine the three‐dimensional structure of the human TBCE, TBCB and &agr;‐tubulin (&agr;EB) complex, which is formed upon &agr;‐tubulin–&bgr;‐tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X‐ray crystallography, allowed description of the molecular architecture of the &agr;EB complex. We found that heterodimer dissociation is an energy‐independent process that takes place through a disruption of the &agr;‐tubulin–&bgr;‐tubulin interface that is caused by a steric interaction between &bgr;‐tubulin and the TBCE cytoskeleton‐associated protein glycine‐rich (CAP‐Gly) and leucine‐rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin‐like (UBL) domains in the &agr;EB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating &agr;‐tubulin degradation.