Elucidating polymorphs of crystal structures by intensity-based hierarchical clustering analysis of multiple diffraction data sets.

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of the diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single-structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference to define the threshold used to group the isomorphous data sets (the `isomorphic threshold') poses a challenge. Here, unit-cell-based and intensity-based HCAs have been applied to data sets for apo trypsin and inhibitor-bound trypsin that were mixed post data acquisition to investigate the efficacy of HCA in classifying polymorphous data sets. Single-step intensity-based HCA successfully classified polymorphs with a certain `isomorphic threshold'. In data sets for several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested `isomorphic threshold'. Polymorphs were also detected in single crystals using data collected using the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis.

Crystal structural analysis of aldoxime dehydratase from Bacillus sp. OxB-1: Importance of surface residues in optimization for crystallization.

Aldoxime dehydratase (Oxd) is a heme enzyme that catalyzes aldoxime dehydration to the corresponding nitriles. Unlike many other heme enzymes, Oxd has a unique feature that the substrate binds directly to the heme. Therefore, it is thought that structural differences around the bound heme directly relate to differences in substrate selection. However sufficient structural information to discuss the substrate specificity has not been obtained. Oxd from Bacillus sp. OxB-1 (OxdB) shows unique substrate specificity and enantioselectivity compared to the Oxds whose crystal structures have already been reported. Here, we report the crystal structure of OxdB, which has not been reported previously. Although the crystallization of OxdB has been difficult, by adding a site-specific mutation to Glu85 located on the surface of the protein, we succeeded in crystallizing OxdB without reducing the enzyme activity. The catalytic triad essential for Oxd activity were structurally conserved in OxdB. In addition, the crystal structure of the Michaelis complex of OxdB and the diastereomerically pure substrate Z-2-(3-bromophenyl)-propanal oxime implied the importance of several hydrophobic residues for substrate specificity. Mutational analysis implicated Ala12 and Ala14 in the E/Z selectivity of bulky compounds. The N-terminal region of OxdB was shown to be shorter than those of Oxds from Pseudomonas chlororaphis and Rhodococcus sp. N-771, and have high flexibility. These structural differences possibly result in distinct preferences for aldoxime substrates based on factors such as substrate size.

Heme controls the structural rearrangement of its sensor protein mediating the hemolytic bacterial survival.

Hemes (iron-porphyrins) are critical for biological processes in all organisms. Hemolytic bacteria survive by acquiring b-type heme from hemoglobin in red blood cells from their animal hosts. These bacteria avoid the cytotoxicity of excess heme during hemolysis by expressing heme-responsive sensor proteins that act as transcriptional factors to regulate the heme efflux system in response to the cellular heme concentration. Here, the underlying regulatory mechanisms were investigated using crystallographic, spectroscopic, and biochemical studies to understand the structural basis of the heme-responsive sensor protein PefR from Streptococcus agalactiae, a causative agent of neonatal life-threatening infections. Structural comparison of heme-free PefR, its complex with a target DNA, and heme-bound PefR revealed that unique heme coordination controls a >20 Å structural rearrangement of the DNA binding domains to dissociate PefR from the target DNA. We also found heme-bound PefR stably binds exogenous ligands, including carbon monoxide, a by-product of the heme degradation reaction.

Use of a Ferritin L134P Mutant for the Facile Conjugation of Prussian Blue in the Apoferritin Cavity.

Since the bullfrog H-ferritin L134P mutant in which leucine 134 is replaced with proline was found to exhibit a flexible conformation in the C3 axis channel, homologous ferritins with the corresponding mutation have often been studied in terms of a mechanism of iron release from the mineral core within the protein cavity. Meanwhile, a ferritin mutant with the flexible channel is an attractive material in developing a method to encapsulate functional molecules larger than mononuclear ions into the protein cavity. This study describes the clathrate with a horse spleen L-ferritin L134P mutant containing Prussian blue (PB) without a frequently used technique, disassembly and reassembly of the protein subunits. The spherical shell of ferritin was confirmed in a TEM image of the clathrate. The produced clathrate (PB@L134P) was soluble in water and reproduced the spectroscopic and electrochemical properties of PB prepared using the conventional method. The catalytic activity for an oxidoreductive reaction with H2O2, one of the major applications of conventional PB, was also observed for the clathrate. The instability of PB in alkaline solutions, limiting its wide applications in aqueous media, was significantly improved in PB@L134P, showing the protective effect of the protein shell. The method developed here shows that horse spleen L-ferritin L134P is a useful scaffold to produce clathrates of three-dimensional complexes with ferritin.

Structural basis for the heme transfer reaction in heme uptake machinery from Corynebacteria.

The crystal structures of the conserved region domains of HtaA and HtaB, which act as heme binding/transport proteins in the heme uptake machinery in Corynebacterium glutamicum, are determined for the first time. The molecular mechanism of heme transfer among these proteins is proposed based on the spectroscopic and structural analyses.

Structural characterization of HypX responsible for CO biosynthesis in the maturation of NiFe-hydrogenase.

Several accessory proteins are required for the assembly of the metal centers in hydrogenases. In NiFe-hydrogenases, CO and CN- are coordinated to the Fe in the NiFe dinuclear cluster of the active center. Though these diatomic ligands are biosynthesized enzymatically, detail mechanisms of their biosynthesis remain unclear. Here, we report the structural characterization of HypX responsible for CO biosynthesis to assemble the active site of NiFe hydrogenase. CoA is constitutionally bound in HypX. Structural characterization of HypX suggests that the formyl-group transfer will take place from N10-formyl-THF to CoA to form formyl-CoA in the N-terminal domain of HypX, followed by decarbonylation of formyl-CoA to produce CO in the C-terminal domain though the direct experimental results are not available yet. The conformation of CoA accommodated in the continuous cavity connecting the N- and C-terminal domains will interconvert between the extended and the folded conformations for HypX catalysis.

Protein Dynamics of the Sensor Protein HemAT as Probed by Time-Resolved Step-Scan FTIR Spectroscopy.

The heme-based aerotactic transducer (HemAT) is an oxygen-sensor protein consisting of a sensor and a signaling domain in the N- and C-terminal regions, respectively. Time-resolved step-scan FTIR spectroscopy was employed to characterize protein intermediate states obtained by photolysis of the carbon monoxide complexes of sensor-domain, full-length HemAT, and the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) HemAT mutants. We assign the spectral components to discrete substructures, which originate from a helical structure that is solvated (1638 cm-1) and a native helix that is protected from solvation by interhelix tertiary interactions (1654 cm-1). The full-length protein is characterized by an additional amide I absorbance at 1661 cm-1, which is attributed to disordered structure suggesting that further protein conformational changes occur in the presence of the signaling domain in the full-length protein. The kinetics monitored within the amide I absorbance of the polypeptide backbone in the sensor domain exhibit two distinct relaxation phases (t1 = 24 and t2 = 694 µs), whereas that of the full-length protein exhibits monophasic behavior for all substructures in a time range of t = 1253-2090 µs. These observations can be instrumental in monitoring helix motion and the role of specific mutants in controlling the dynamics in the communication pathway from the sensor to the signaling domain. The kinetics observed for the amide I relaxation for the full-length protein indicate that the discrete substructures within full-length HemAT, unlike those of the sensor domain, relax independently.

Probing the Role of the Heme Distal and Proximal Environment in Ligand Dynamics in the Signal Transducer Protein HemAT by Time-Resolved Step-Scan FTIR and Resonance Raman Spectroscopy.

HemAT is a heme-containing oxygen sensor protein that controls aerotaxis. Time-resolved step-scan FTIR studies were performed on the isolated sensor domain and full-length HemAT proteins as well as on the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) mutants to elucidate the effect of the site-specific mutations on the ligand dynamics subsequent to CO photolysis. The mutations aimed to perturb H-bonding and electrostatic interactions near the heme Fe-bound gaseous ligand (CO) and the heme proximal environment. Rebinding of CO to the heme Fe is biphasic in the sensor domain and full-length HemAT as well as in the mutants, with the exception of the Y133F mutant protein. The monophasic rebinding of CO in Y133F suggests that in the absence of the H-bond between Y133 and the heme proximal H123 residue the ligand rebinding process is significantly affected. The role of the proximal environment is also probed by resonance Raman photodissociation experiments, in which the Fe-His mode of the photoproduct of sensor domain HemAT-CO is detected at a frequency higher than that of the deoxy form in the difference resonance Raman spectra. The role of the conformational changes of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in regulating ligand dynamics in the heme pocket are discussed.

Visible-light-induced release of CO by thiolate iron(iii) carbonyl complexes bearing N,C,S-pincer ligands.

Iron(iii) carbonyl complexes are stabilized by a pincer ligand containing pyridine-N, phenyl-C and thiolate-S donors and two axial phosphine ligands. The N,C,S-pincer iron(iii) carbonyl complexes show CO-releasing properties induced by visible light.

A Study of the Dynamics of the Heme Pocket and C-helix in CooA upon CO Dissociation Using Time-Resolved Visible and UV Resonance Raman Spectroscopy.

CooA is a CO-sensing transcriptional activator from the photosynthetic bacterium Rhodospirillum rubrum that binds CO at the heme iron. The heme iron in ferrous CooA has two axial ligands: His77 and Pro2. CO displaces Pro2 and induces a conformational change in CooA. The dissociation of CO and/or ligation of the Pro2 residue are believed to trigger structural changes in the protein. Visible time-resolved resonance Raman spectra obtained in this study indicated that the ν(Fe-His) mode, arising from the proximal His77-iron stretch, does not shift until 50 µs after the photodissociation of CO. Ligation of the Pro2 residue to the heme iron was observed around 50 µs after the photodissociation of CO, suggesting that the ν(Fe-His) band exhibits no shift until the ligation of Pro2. UV resonance Raman spectra suggested structural changes in the vicinity of Trp110 in the C-helix upon CO binding, but no or very small spectral changes in the time-resolved UV resonance Raman spectra were observed from 100 ns to 100 µs after the photodissociation of CO. These results strongly suggest that the conformational change of CooA is induced by the ligation of Pro2 to the heme iron.

Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters.

Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.

Identification of essential histidine residues involved in heme binding and Hemozoin formation in heme detoxification protein from Plasmodium falciparum.

Malaria parasites digest hemoglobin within a food vacuole to supply amino acids, releasing the toxic product heme. During the detoxification, toxic free heme is converted into an insoluble crystalline form called hemozoin (Hz). Heme detoxification protein (HDP) in Plasmodium falciparum is one of the most potent of the hemozoin-producing enzymes. However, the reaction mechanisms of HDP are poorly understood. We identified the active site residues in HDP using a combination of Hz formation assay and spectroscopic characterization of mutant proteins. Replacement of the critical histidine residues His122, His172, His175, and His197 resulted in a reduction in the Hz formation activity to approximately 50% of the wild-type protein. Spectroscopic characterization of histidine-substituted mutants revealed that His122 binds heme and that His172 and His175 form a part of another heme-binding site. Our results show that the histidine residues could be present in the individual active sites and could be ligated to each heme. The interaction between heme and the histidine residues would serve as a molecular tether, allowing the proper positioning of two hemes to enable heme dimer formation. The heme dimer would act as a seed for the crystal growth of Hz in P. falciparum.

Heme-binding properties of heme detoxification protein from Plasmodium falciparum.

The heme detoxification protein of the malaria parasite Plasmodium falciparum is involved in the formation of hemozoin, an insoluble crystalline form of heme. Although the disruption of hemozoin formation is the most widely used strategy for controlling the malaria parasite, the heme-binding properties of heme detoxification protein are poorly characterized. In this study, we established a method for the expression and purification of the non-tagged protein and characterized heme-binding properties. The spectroscopic features of non-tagged protein differ from those of the His-tagged protein, suggesting that the artificial tag interferes with the properties of the recombinant protein. The purified recombinant non-tagged heme detoxification protein had two heme-binding sites and exhibited a spectrum typical of heme proteins. A mechanism for hemozoin formation is proposed.

The Dos family of globin-related sensors using PAS domains to accommodate haem acting as the active site for sensing external signals.

Sensor proteins play crucial roles in maintaining homeostasis of cells by sensing changes in extra- and intracellular chemical and physical conditions to trigger biological responses. It has recently become clear that gas molecules function as signalling molecules in these biological regulatory systems responsible for transcription, chemotaxis, synthesis/hydrolysis of nucleotide second messengers, and other complex physiological processes. Haem-containing sensor proteins are widely used to sense gas molecules because haem can bind gas molecules reversibly. Ligand binding to the haem in the sensor proteins triggers conformational changes around the haem, which results in their functional regulation. Spectroscopic and crystallographic studies are essential to understand how these sensor proteins function in these biological regulatory systems. In this chapter, I discuss structural and functional relationships of haem-containing PAS and PAS-related families of the sensor proteins.

A model theoretical study on ligand exchange reactions of CooA.

Rr-CooA is a CO-sensor heme protein, where binding of CO with the heme group stimulates a transcriptional activator activity of CooA. In this process, the heme undergoes a series of ligand exchanges. In the ferric form, the heme has Cys75 and Pro2 as the axial ligands. In the reduced ferrous form, the heme has His77 instead of Cys75 as an axial ligand with Pro2. Only in the reduced form, CooA can bind CO that replaces Pro2. Model calculations are carried out to elucidate the ligand exchange reactions of CooA. The coordinated proline is found to be the neutral, protonated form. The ligand exchange of cysteine for histidine is reproduced by a relatively small model. This exchange would be mainly due to difference in stability of the non-bonding sulfur p-orbital in Cys75 between the ferric and ferrous states. The selectivity of gas molecules among CO, NO, and O2 in the proteins is explained by the relative stability of products for Rr-CooA. This is also the case for Ch-CooA, where the amino group of the N-terminus and a histidine are coordinated to the iron ion both in the ferric and ferrous states. The ability to bind the gas molecules is a little stronger in Rr-CooA than in Ch-CooA. In the ferric form of Rr-CooA, heme is deformed to a ruffled form whereas heme is planar in the ferrous form, which leads to a red-shifted Q-band in the former.

Structural basis for the transcriptional regulation of heme homeostasis in Lactococcus lactis.

Although heme is a crucial element for many biological processes including respiration, heme homeostasis should be regulated strictly due to the cytotoxicity of free heme molecules. Numerous lactic acid bacteria, including Lactococcus lactis, acquire heme molecules exogenously to establish an aerobic respiratory chain. A heme efflux system plays an important role for heme homeostasis to avoid cytotoxicity of acquired free heme, but its regulatory mechanism is not clear. Here, we report that the transcriptional regulator heme-regulated transporter regulator (HrtR) senses and binds a heme molecule as its physiological effector to regulate the expression of the heme-efflux system responsible for heme homeostasis in L. lactis. To elucidate the molecular mechanisms of how HrtR senses a heme molecule and regulates gene expression for the heme efflux system, we determined the crystal structures of the apo-HrtR·DNA complex, apo-HrtR, and holo-HrtR at a resolution of 2.0, 3.1, and 1.9 Å, respectively. These structures revealed that HrtR is a member of the TetR family of transcriptional regulators. The residue pair Arg-46 and Tyr-50 plays a crucial role for specific DNA binding through hydrogen bonding and a CH-π interaction with the DNA bases. HrtR adopts a unique mechanism for its functional regulation upon heme sensing. Heme binding to HrtR causes a coil-to-helix transition of the α4 helix in the heme-sensing domain, which triggers a structural change of HrtR, causing it to dissociate from the target DNA for derepression of the genes encoding the heme efflux system. HrtR uses a unique heme-sensing motif with bis-His (His-72 and His-149) ligation to the heme, which is essential for the coil-to-helix transition of the α4 helix upon heme sensing.

Structural basis for oxygen sensing and signal transduction of the heme-based sensor protein Aer2 from Pseudomonas aeruginosa.

The crystal structure of a truncated Aer2, a signal transducer protein from Pseudomonas aeruginosa, consisting of the heme-containing PAS and di-HAMP domains revealed that a distal tryptophan residue (Trp283) plays an important role in stabilizing the heme-bound O(2) and intra-molecular signal transduction upon O(2) binding.

Structural dynamics of proximal heme pocket in HemAT-Bs associated with oxygen dissociation.

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-containing O(2) sensor protein that acts as a chemotactic signal transducer. Binding of O(2) to the heme in the sensor domain of HemAT-Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O(2)-dependent conformational changes in HemAT-Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT-Bs upon O(2) and CO dissociation. A comparison between the O(2) and CO complexes provides insights on O(2)/CO discrimination in HemAT-Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10ns and 100µs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe-His), for the O(2)-dissociated HemAT-Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O(2) dissociation would be associated with the O(2)/CO discrimination in HemAT-Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT-Bs, which showed that the structural dynamics related to O(2) dissociation for the full-length HemAT-Bs are faster than those for the sensor domain HemAT-Bs. This indicates that the heme proximal structural dynamics upon O(2) dissociation are coupled with signal transduction in HemAT-Bs.

Site-specific protein dynamics in communication pathway from sensor to signaling domain of oxygen sensor protein, HemAT-Bs: Time-resolved Ultraviolet Resonance Raman Study.

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ∼50 µs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ∼50 µs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 µs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.

Novel bacterial gas sensor proteins with transition metal-containing prosthetic groups as active sites.

SIGNIFICANCE: Gas molecules function as signaling molecules in many biological regulatory systems responsible for transcription, chemotaxis, and other complex physiological processes. Gas sensor proteins play a crucial role in regulating such biological systems in response to gas molecules. RECENT ADVANCES: New sensor proteins that sense oxygen or nitric oxide have recently been found, and they have been characterized by X-ray crystallographic and/or spectroscopic analysis. It has become clear that the interaction between a prosthetic group and gas molecules triggers dynamic structural changes in the protein backbone when a gas sensor protein senses gas molecules. Gas sensor proteins employ novel mechanisms to trigger conformational changes in the presence of a gas. CRITICAL ISSUES: In gas sensor proteins that have iron-sulfur clusters as active sites, the iron-sulfur clusters undergo structural changes, which trigger a conformational change. Heme-based gas sensor proteins reconstruct hydrogen-bonding networks around the heme and heme-bound ligand. FUTURE DIRECTION: Gas sensor proteins have two functional states, on and off, which are active and inactive, respectively, for subsequent signal transduction in response to their physiological effector molecules. To fully understand the structure-function relationships of gas sensor proteins, it is vital to perform X-ray crystal structure analyses of full-length proteins in both the on and off states.