William R. Montfort

Crystal structure and electron transfer kinetics of CueO, a multicopper oxidase required for copper homeostasis in Escherichia coli

CueO (YacK), a multicopper oxidase, is part of the copper-regulatory cue operon in Escherichia coli. The crystal structure of CueO has been determined to 1.4-Å resolution by using multiple anomalous dispersion phasing and an automated building procedure that yielded a nearly complete model without manual intervention. This is the highest resolution multicopper oxidase structure yet determined and provides a particularly clear view of the four coppers at the catalytic center. The overall structure is similar to those of laccase and ascorbate oxidase, but contains an extra 42-residue insert in domain 3 that includes 14 methionines, nine of which lie in a helix that covers the entrance to the type I (T1, blue) copper site. The trinuclear copper cluster has a conformation not previously seen: the Cu-O-Cu binuclear species is nearly linear (Cu-O-Cu bond angle = 170°) and the third (type II) copper lies only 3.1 Å from the bridging oxygen. CueO activity was maximal at pH 6.5 and in the presence of >100 μM Cu(II). Measurements of intermolecular and intramolecular electron transfer with laser flash photolysis in the absence of Cu(II) show that, in addition to the normal reduction of the T1 copper, which occurs with a slow rate (k = 4 × 107 M−1⋅s−1), a second electron transfer process occurs to an unknown site, possibly the trinuclear cluster, with k = 9 × 107 M−1⋅s−1, followed by a slow intramolecular electron transfer to T1 copper (k ∼10 s−1). These results suggest the methionine-rich helix blocks access to the T1 site in the absence of excess copper.

Cuprous oxidase activity of CueO from Escherichia coli.

We have found CueO from Escherichia coli to have a robust cuprous oxidase activity, severalfold higher than any homologue. These data suggest that a functional role for CueO in protecting against copper toxicity in vivo includes the removal of Cu(I).

Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial

The nitrophorins comprise an unusual family of proteins that use ferric (Fe(III)) heme to transport highly reactive nitric oxide (NO) from the salivary gland of a blood sucking bug to the victim, resulting in vasodilation and reduced blood coagulation. We have determined structures of nitrophorin 4 in complexes with H2O, cyanide and nitric oxide. These structures reveal a remarkable feature: the nitrophorins have a broadly open distal pocket in the absence of NO, but upon NO binding, three or more water molecules are expelled and two loops fold into the distal pocket, resulting in the packing of hydrophobic groups around the NO molecule and increased distortion of the heme. In this way, the protein apparently forms a ‘hydrophobic trap’ for the NO molecule. The structures are very accurate, ranging between 1.6 and 1.4 Å resolutions.

Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds

Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a “stepping-stone” method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and λ. We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and λ. The domains show 40% sequence identity but differ by switching of α-helix to β-sheet in a C-terminal region spanning ≈25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization.

S-nitrosylation-induced conformational change in blackfin tuna myoglobin.

S-Nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by “wedging” apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-Å structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

Crystal Structure of the Bacterial Luciferase/Flavin Complex Provides Insight into the Function of the β Subunit

Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

Crystal structure of human thioredoxin revealing an unraveled helix and exposed S-nitrosation site

Thioredoxins reduce disulfide bonds and other thiol modifications in all cells using a CXXC motif. Human thioredoxin 1 is unusual in that it codes for an additional three cysteines in its 105 amino acid sequence, each of which have been implicated in other reductive activities. Cys 62 and Cys 69 are buried in the protein interior and lie at either end of a short helix (helix 3), and yet can disulfide link under oxidizing conditions. Cys 62 is readily S‐nitrosated, giving rise to a SNO modification, which is also buried. Here, we present two crystal structures of the C69S/C73S mutant protein under oxidizing (1.5 Å) and reducing (1.1 Å) conditions. In the oxidized structure, helix 3 is unraveled and displays a new conformation that is stabilized by a series of new hydrogen bonds and a disulfide link with Cys 62 in a neighboring molecule. The new conformation provides an explanation for how a completely buried residue can participate in SNO exchange reactions.

Binding of YC-1 or BAY 41-2272 to Soluble Guanylyl Cyclase Induces a Geminate Phase in CO Photolysis

Soluble guanylyl/guanylate cyclase (sGC), a heme-containing heterodimeric protein of approximately 150 kDa, is the primary receptor for nitric oxide, an endogenous molecule of immense physiological importance to animals. Recent studies have identified compounds such as YC-1 and BAY 41-2272 that stimulate sGC independently of NO binding, properties of importance for the treatment of endothelial dysfunction and other diseases linked to malfunctioning NO signaling pathways. We have developed a novel expression system for sGC from Manduca sexta (the tobacco hornworm) that retains the N-terminal two-thirds of both subunits, including heme, but is missing the catalytic domain. Here, we show that binding of compounds YC-1 or BAY 41-2272 to the truncated protein leads to a change in the heme pocket such that photolyzed CO cannot readily escape from the protein matrix. Geminate recombination of the trapped CO molecules with heme takes place with a measured rate of 6 x 10(7) s(-1). These findings provide strong support for an allosteric regulatory model in which YC-1 and related compounds can alter the sGC heme pocket conformation to retain diatomic ligands and thus activate the enzyme alone or in synergy with either NO or CO.

N15 Cro and λ Cro: Orthologous DNA-binding domains with completely different but equally effective homodimer interfaces

Bacteriophage Cro proteins bind to target DNA as dimers but do not all dimerize with equal strength, and differ in fold in the region of the dimer interface. We report the structure of the Cro protein from Enterobacteria phage N15 at 1.05 Å resolution. The subunit fold contains five α‐helices and is closely similar to the structure of P22 Cro (1.3 Å backbone room mean square difference over 52 residues), but quite different from that of λ Cro, a structurally diverged member of this family with a mixed α‐helix/β‐sheet fold. N15 Cro crystallizes as a biological dimer with an extensive interface (1303 Å2 change in accessible surface area per dimer) and also dimerizes in solution with a Kd of 5.1 ± 1.5 μM. Its dimerization is much stronger than that of its structural homolog P22 Cro, which does not self‐associate detectably in solution. Instead, the level of self‐association and interfacial area for N15 Cro is similar to that of λ Cro, even though these two orthologs do not share the same fold and have dimer interfaces that are qualitatively different in structure. The common Cro ancestor is thought to be an all‐helical monomer similar to P22 Cro. We propose that two Cro descendants independently developed stronger dimerization by entirely different mechanisms.

Apo-nitrophorin 4 at atomic resolution.

The nitrophorins from Rhodnius prolixus, the kissing bug, are heme‐containing proteins used for the transport of nitric oxide to aide the insect in obtaining a blood meal. The Rhodnius nitrophorins display an eight‐stranded antiparallel beta‐barrel motif, typical of lipocalins, with a histidine‐linked heme in the open end of the barrel. Heme is stabilized in the ferric state and highly distorted, displaying a ruffled conformation that may be of importance in the setting of the reduction potential. To help in understanding the means by which the protein matrix, an inherently soft material, is able to distort the heme from its low‐energy planar conformation, we have determined the crystal structure of apo‐nitrophorin 4–1.1 Å resolution. Removal of the heme from nitrophorin 4 has very little effect on its structure: The heme binding cavity remains open and the loops near the cavity entrance respond to lower pH in the same manner as the intact protein. We conclude that the general stability of the lipocalin fold and apparent rigidity of the beta‐barrel provide the means for distorting the heme cofactor.