James A. Endrizzi

Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases.

A family of eukaryotic-like Ser/Thr protein kinases occurs in bacteria, but little is known about the structures and functions of these proteins. Here we characterize PknB, a transmembrane signaling kinase from Mycobacterium tuberculosis. The intracellular PknB kinase domain is active autonomously, and the active enzyme is phosphorylated on residues homologous to regulatory phospho-acceptors in eukaryotic Ser/Thr kinases. The crystal structure of the PknB kinase domain in complex with an ATP analog reveals the active conformation. The predicted fold of the PknB extracellular domain matches the proposed targeting domain of penicillin-binding protein 2x. The structural and chemical similarities of PknB to metazoan homologs support a universal activation mechanism of Ser/Thr protein kinases in prokaryotes and eukaryotes.

Crystal structure of E. coli β–carbonic anhydrase, an enzyme with an unusual pH–dependent activity

Carbonic anhydrases fall into three distinct evolutionary and structural classes: α, β, and γ. The β‐class carbonic anhydrases (β‐CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a β‐CA from Escherichia coli, to a resolution of 2.0 Å. In agreement with the structure of the β‐CA from the chloroplast of the red alga Porphyridium purpureum, the active‐site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some β‐CAs. The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH‐dependent in the physiological range, and its expression in yeast complements an oxygen‐sensitive phenotype displayed by a β‐CA‐deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other β‐CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.

Structural basis of dimerization, coactivator recognition and MODY3 mutations in HNF-1alpha.

Maturity-onset diabetes of the young type 3 (MODY3) results from mutations in the transcriptional activator hepatocyte nuclear factor-1α (HNF-1α). Several MODY3 mutations target the HNF-1α dimerization domain (HNF-p1), which binds the coactivator, dimerization cofactor of HNF-1 (DCoH). To define the mechanism of coactivator recognition and the basis for the MODY3 phenotype, we determined the cocrystal structure of the DCoH–HNF-p1 complex and characterized biochemically the effects of MODY3 mutations in HNF-p1. The DCoH–HNF-p1 complex comprises a dimer of dimers in which HNF-p1 forms a unique four-helix bundle. Through rearrangements of interfacial side chains, a single, bifunctional interface in the DCoH dimer mediates both HNF-1α binding and formation of a competing, transcriptionally inactive DCoH homotetramer. Consistent with the structure, MODY3 mutations in HNF-p1 reduce activator function by two distinct mechanisms.

High-resolution structure of the HNF-1alpha dimerization domain.

The N-terminal dimerization domain of the transcriptional activator hepatocyte nuclear factor-1alpha (HNF-1alpha) is essential for DNA binding and association of the transcriptional coactivator, DCoH (dimerization cofactor of HNF-1). To investigate the basis for dimerization of HNF-1 proteins, we determined the 1.2 A resolution X-ray crystal structure of the dimerization domain of HNF-1alpha (HNF-p1). Phasing was facilitated by devising a simple synthesis for Fmoc-selenomethionine and substituting leucine residues with selenomethionine. The HNF-1 dimerization domain forms a unique, four-helix bundle that is preserved with localized conformational shifts in the DCoH complex. In three different crystal forms, HNF-p1 displays subtle shifts in the conformation of the interhelix loop and the crossing angle between the amino- and carboxyl-terminal helices. In all three crystal forms, the HNF-p1 dimers pair through an exposed hydrophobic surface that also forms the binding site for DCoH. Conserved core residues in the dimerization domain of the homologous transcriptional regulator HNF-1beta rationalize the functional heterodimerization of the HNF-1alpha and HNF-1beta proteins. Mutations in HNF-1alpha are associated with maturity-onset diabetes of the young type 3 (MODY3), and the structure of HNF-p1 provides insights into the effects of three MODY3 mutations.

Human CTP synthase filament structure reveals the active enzyme conformation

The universally conserved enzyme CTP synthase (CTPS) forms filaments in bacteria and eukaryotes. In bacteria, polymerization inhibits CTPS activity and is required for nucleotide homeostasis. Here we show that for human CTPS, polymerization increases catalytic activity. The cryo-EM structures of bacterial and human CTPS filaments differ considerably in overall architecture and in the conformation of the CTPS protomer, explaining the divergent consequences of polymerization on activity. The structure of human CTPS filament, the first structure of the full-length human enzyme, reveals a novel active conformation. The filament structures elucidate allosteric mechanisms of assembly and regulation that rely on a conserved conformational equilibrium. The findings may provide a mechanism for increasing human CTPS activity in response to metabolic state and challenge the assumption that metabolic filaments are generally storage forms of inactive enzymes. Allosteric regulation of CTPS polymerization by ligands likely represents a fundamental mechanism underlying assembly of other metabolic filaments.

Cloning, crystallization and preliminary characterization of a β-carbonic anhydrase from Escherichia coli

Carbonic anhydrases are zinc metalloenzymes that fall into three distinct evolutionary and structural classes, alpha, beta and gamma. Although alpha-class enzymes, particularly mammalian carbonic anhydrase II, have been the subject of extensive structural studies, for the beta class, consisting of a wide variety of prokaryotic and plant chloroplast carbonic anhydrases, the structural data is quite limited. A member of the beta class from E. coli (CynT2) has been crystallized in native and selenomethionine-labelled forms and multiwavelength anomalous dispersion techniques have been applied in order to determine the positions of anomalous scatterers. The resulting phase information is sufficient to produce an interpretable electron-density map. A crystal structure for CynT2 would contribute significantly to the emerging structural knowledge of a biologically important class of enzymes that perform critical functions in carbon fixation and prokaryotic metabolism.

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non‐polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild‐type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate‐analog bound conformation and the third hinge angle is intermediate to the other two. The active‐site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active‐site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active‐site conformations.

Binding of bisubstrate analog promotes large structural changes in the unregulated catalytic trimer of aspartate transcarbamoylase: Implications for allosteric regulation

A central problem in understanding enzyme regulation is to define the conformational states that account for allosteric changes in catalytic activity. ForEscherichia coli aspartate transcarbamoylase (ATCase; EC 2.1.3.2) the active, relaxed (R state) holoenzyme is generally assumed to be represented by the crystal structure of the complex of the holoenzyme with the bisubstrate analogN-phosphonacetyl-L-aspartate (PALA). It is unclear, however, which conformational differences between the unliganded, inactive, taut (T state) holoenzyme and the PALA complex are attributable to localized effects of inhibitor binding as contrasted to the allosteric transition. To define the conformational changes in the isolated, nonallosteric C trimer resulting from the binding of PALA, we determined the 1.95-Å resolution crystal structure of the C trimer-PALA complex. In contrast to the free C trimer, the PALA-bound trimer exhibits approximate threefold symmetry. Conformational changes in the C trimer upon PALA binding include ordering of two active site loops and closure of the hinge relating the N- and C-terminal domains. The C trimer-PALA structure closely resembles the liganded C subunits in the PALA-bound holoenzyme. This similarity suggests that the pronounced hinge closure and other changes promoted by PALA binding to the holoenzyme are stabilized by ligand binding. Consequently, the conformational changes attributable to the allosteric transition of the holoenzyme remain to be defined.