Eduard Bitto

Crystallographic Structure of SurA, a Molecular Chaperone that Facilitates Folding of Outer Membrane Porins

The SurA protein facilitates correct folding of outer membrane proteins in gram-negative bacteria. The sequence of Escherichia coli SurA presents four segments, two of which are peptidyl-prolyl isomerases (PPIases); the crystal structure reveals an asymmetric dumbbell, in which the amino-terminal, carboxy-terminal, and first PPIase segments of the sequence form a core structural module, and the second PPIase segment is a satellite domain tethered approximately 30 A from this module. The core module, which is implicated in membrane protein folding, has a novel fold that includes an extended crevice. Crystal contacts show that peptides bind within the crevice, suggesting a model for chaperone activity whereby segments of polypeptide may be repetitively sequestered and released during the membrane protein-folding process.

Structure of aspartoacylase, the brain enzyme impaired in Canavan disease

Aspartoacylase catalyzes hydrolysis of N-acetyl-l-aspartate to aspartate and acetate in the vertebrate brain. Deficiency in this activity leads to spongiform degeneration of the white matter of the brain and is the established cause of Canavan disease, a fatal progressive leukodystrophy affecting young children. We present crystal structures of recombinant human and rat aspartoacylase refined to 2.8- and 1.8-Å resolution, respectively. The structures revealed that the N-terminal domain of aspartoacylase adopts a protein fold similar to that of zinc-dependent hydrolases related to carboxypeptidases A. The catalytic site of aspartoacylase shows close structural similarity to those of carboxypeptidases despite only 10–13% sequence identity between these proteins. About 100 C-terminal residues of aspartoacylase form a globular domain with a two-stranded β-sheet linker that wraps around the N-terminal domain. The long channel leading to the active site is formed by the interface of the N- and C-terminal domains. The C-terminal domain is positioned in a way that prevents productive binding of polypetides in the active site. The structures revealed that residues 158–164 may undergo a conformational change that results in opening and partial closing of the channel entrance. We hypothesize that the catalytic mechanism of aspartoacylase is closely analogous to that of carboxypeptidases. We identify residues involved in zinc coordination, and propose which residues may be involved in substrate binding and catalysis. The structures also provide a structural framework necessary for understanding the deleterious effects of many missense mutations of human aspartoacylase.

Binding of phage-display-selected peptides to the periplasmic chaperone protein SurA mimics binding of unfolded outer membrane proteins

SurA is a periplasmic chaperone protein that facilitates maturation of integral outer membrane proteins (OMPs). Short peptides that bind SurA have previously been characterized. In this work, an enzyme‐linked immunoabsorbent assay‐based competition assay is utilized to demonstrate that binding of such peptides, presented by peptide‐tagged phage, mimics binding of biological substrates. Two representative unfolded OMPs, OmpF and OmpG, bind SurA and a core structural fragment thereof in competition with peptide‐tagged phage, and with the same order‐of‐magnitude affinity as the peptides. Additionally, unfolded OmpF and OmpG bind SurA more tightly than an unfolded water‐soluble protein, while folded proteins have no measurable affinity, demonstrating a specificity of SurA for OMP polypeptides.

X-RAY STRUCTURE OF ILL2, AN AUXIN-CONJUGATE AMIDOHYDROLASE FROM ARABIDOPSIS THALIANA

The plant hormone indole‐3‐acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X‐ray crystal structure of IAA‐amino acid hydrolase IAA‐leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 Å resolution. ILL2 preferentially hydrolyses the auxin‐amino acid conjugate N‐(indol‐3‐acetyl)‐alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three‐layer αβα sandwich architecture and aminopeptidase topology and a smaller satellite domain with two‐layer αβ‐sandwich architecture and α–β‐plaits topology. The metal‐coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N‐(indol‐3‐acetyl)‐alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side‐chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid‐base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family. Proteins 2009.

Structure of an ETHE1-like protein from Arabidopsis thaliana.

The protein product of gene At1g53580 from Arabidopsis thaliana possesses 54% sequence identity to a human enzyme that has been implicated in the rare disorder ethylmalonic encephalopathy. The structure of the At1g53580 protein has been solved to a nominal resolution of 1.48 Angstrom. This structure reveals tertiary structure differences between the ETHE1-like enzyme and glyoxalase II enzymes that are likely to account for differences in reaction chemistry and multimeric state between the two types of enzymes. In addition, the Arabidopsis ETHE1 protein is used as a model to explain the significance of several mutations in the human enzyme that have been observed in patients with ethylmalonic encephalopathy.

Structure of Human J-type Co-chaperone HscB Reveals a Tetracysteine Metal-binding Domain

Iron-sulfur proteins play indispensable roles in a broad range of biochemical processes. The biogenesis of iron-sulfur proteins is a complex process that has become a subject of extensive research. The final step of iron-sulfur protein assembly involves transfer of an iron-sulfur cluster from a cluster-donor to a cluster-acceptor protein. This process is facilitated by a specialized chaperone system, which consists of a molecular chaperone from the Hsc70 family and a co-chaperone of the J-domain family. The 3.0Å crystal structure of a human mitochondrial J-type co-chaperone HscB revealed an L-shaped protein that resembles Escherichia coli HscB. The important difference between the two homologs is the presence of an auxiliary metal-binding domain at the N terminus of human HscB that coordinates a metal via the tetracysteine consensus motif CWXCX9–13FCXXCXXXQ. The domain is found in HscB homologs from animals and plants as well as in magnetotactic bacteria. The metal-binding site of the domain is structurally similar to that of rubredoxin and several zinc finger proteins containing rubredoxin-like knuckles. The normal mode analysis of HscB revealed that this L-shaped protein preferentially undergoes a scissors-like motion that correlates well with the conformational changes of human HscB observed in the crystals.

Structure of Pyrimidine 5′-Nucleotidase Type 1 INSIGHT INTO MECHANISM OF ACTION AND INHIBITION DURING LEAD POISONING

Eukaryotic pyrimidine 5′-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5′-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35Å resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25Å, 2) phosphoenzyme intermediate analog at 2.30Å, 3) product-transition complex analog at 2.35Å, and 4) product complex at 2.1Å resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35Å) revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides.

Creating protein models from electron-density maps using particle-filtering methods

MOTIVATION One bottleneck in high-throughput protein crystallography is interpreting an electron-density map, that is, fitting a molecular model to the 3D picture crystallography produces. Previously, we developed ACMI (Automatic Crystallographic Map Interpreter), an algorithm that uses a probabilistic model to infer an accurate protein backbone layout. Here, we use a sampling method known as particle filtering to produce a set of all-atom protein models. We use the output of ACMI to guide the particle filters sampling, producing an accurate, physically feasible set of structures. RESULTS We test our algorithm on 10 poor-quality experimental density maps. We show that particle filtering produces accurate all-atom models, resulting in fewer chains, lower sidechain RMS error and reduced R factor, compared to simply placing the best-matching sidechains on ACMIs trace. We show that our approach produces a more accurate model than three leading methods--Textal, Resolve and ARP/WARP--in terms of main chain completeness, sidechain identification and crystallographic R factor. AVAILABILITY Source code and experimental density maps available at http://ftp.cs.wisc.edu/machine-learning/shavlik-group/programs/acmi/

The structure and NO binding properties of the nitrophorin‐like heme‐binding protein from Arabidopsis thaliana gene locus At1g79260.1

The protein from Arabidopsis thaliana gene locus At1g79260.1 is comprised of 166‐residues and is of previously unknown function. Initial structural studies by the Center for Eukaryotic Structural Genomics (CESG) suggested that this protein might bind heme, and consequently, the crystal structures of apo and heme‐bound forms were solved to near atomic resolution of 1.32 Å and 1.36 Å, respectively. The rate of hemin loss from the protein was measured to be 3.6 × 10−5 s−1, demonstrating that it binds heme specifically and with high affinity. The protein forms a compact 10‐stranded β‐barrel that is structurally similar to the lipocalins and fatty acid binding proteins (FABPs). One group of lipocalins, the nitrophorins (NP), are heme proteins involved in nitric oxide (NO) transport and show both sequence and structural similarity to the protein from At1g79260.1 and two human homologues, all of which contain a proximal histidine capable of coordinating a heme iron. Rapid‐mixing and laser photolysis techniques were used to determine the rate constants for carbon monoxide (CO) binding to the ferrous form of the protein (k′CO = 0.23 μM−1 s−1, kCO = 0.050 s−1) and NO binding to the ferric form (k′NO = 1.2 μM–1 s–1, kNO = 73 s−1). Based on both structural and functional similarity to the nitrophorins, we have named the protein nitrobindin and hypothesized that it plays a role in NO transport. However, one of the two human homologs of nitrobindin contains a THAP domain, implying a possible role in apoptosis. Proteins 2010.

X-ray structure of Danio rerio secretagogin: A hexa-EF-hand calcium sensor.

Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF‐hand proteins represent a superfamily of calcium‐binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa‐EF‐hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X‐ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium‐free form at 2.1‐Å resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF‐hand motifs. The domains are arranged into a V‐shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium‐loaded calbindin D28K revealed a striking difference in the spatial arrangement of their domains, which involves ∼180° rotation of the first globular domain with respect to the module formed by the remaining domains. Proteins 2009.