Mikalai Lapkouski

Structure of the motor subunit of type I restriction-modification complex EcoR124I

Type I restriction-modification enzymes act as conventional adenine methylases on hemimethylated DNAs, but unmethylated recognition targets induce them to translocate thousands of base pairs before cleaving distant sites nonspecifically. The first crystal structure of a type I motor subunit responsible for translocation and cleavage suggests how the pentameric translocating complex is assembled and provides a structural framework for translocation of duplex DNA by RecA-like ATPase motors.

Raman Spectroscopy Adds Complementary Detail to the High-Resolution X-Ray Crystal Structure of Photosynthetic Psbp from Spinacia Oleracea.

Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally “dry,” has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.

Crystallization and preliminary crystallographic characterization of the extrinsic PsbP protein of photosystem II from Spinacia oleracea

Preliminary X-ray diffraction analysis of the extrinsic PsbP protein of photosystem II from spinach (Spinacia oleracea) was performed using N-terminally His-tagged recombinant PsbP protein overexpressed in Escherichia coli. Recombinant PsbP protein (thrombin-digested recombinant His-tagged PsbP) stored in bis-Tris buffer pH 6.00 was crystallized using the sitting-drop vapour-diffusion technique with PEG 550 MME as a precipitant and zinc sulfate as an additive. SDS-PAGE analysis of a dissolved crystal showed that the crystals did not contain the degradation products of recombinant PsbP protein. PsbP crystals diffracted to 2.06 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 38.68, b = 46.73, c = 88.9 A.

Structural organization of WrbA in apo- and holoprotein crystals☆

Two previously reported holoprotein crystal forms of the flavodoxin-like E. coli protein WrbA, diffracting to 2.6 and 2.0 A resolution, and new crystals of WrbA apoprotein diffracting to 1.85 A, are refined and analysed comparatively through the lens of flavodoxin structures. The results indicate that differences between apo- and holoWrbA crystal structures are manifested on many levels of protein organization as well as in the FMN-binding sites. Evaluation of the influence of crystal contacts by comparison of lattice packing reveals the proteins global response to FMN binding. Structural changes upon cofactor binding are compared with the monomeric flavodoxins. Topologically non-equivalent residues undergo remarkably similar local structural changes upon FMN binding to WrbA or to flavodoxin, despite differences in multimeric organization and residue types at the binding sites. Analysis of the three crystal structures described here, together with flavodoxin structures, rationalizes functional similarities and differences of the WrbAs relative to flavodoxins, leading to a new understanding of the defining features of WrbAs. The results suggest that WrbAs are not a remote and unusual branch of the flavodoxin family as previously thought but rather a central member with unifying structural features.

Functional Coupling of Duplex Translocation to DNA Cleavage in a Type I Restriction Enzyme.

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.

1.2 Å resolution crystal structure of Escherichia coli WrbA holoprotein

The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Å resolution and are not analysed further here. The 1.2 Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.

Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis

The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 A, respectively.

Purification, crystallization and preliminary X-ray analysis of the HsdR subunit of the EcoR124I endonuclease from Escherichia coli.

EcoR124I is a multicomplex enzyme belonging to the type I restriction-modification system from Escherichia coli. Although EcoR124I has been extensively characterized biochemically, there is no direct structural information available about particular subunits. HsdR is a motor subunit that is responsible for ATP hydrolysis, DNA translocation and cleavage of the DNA substrate recognized by the complex. Recombinant HsdR subunit was crystallized using the sitting-drop vapour-diffusion method. Crystals belong to the primitive monoclinic space group, with unit-cell parameters a = 85.75, b = 124.71, c = 128.37 A, beta = 108.14 degrees. Native data were collected to 2.6 A resolution at the X12 beamline of EMBL Hamburg.

Expression, purification and preliminary crystallization study of RpaC protein from Synechocystis sp. PCC6803

State transitions in cyanobacteria are physiological adaptation mechanisms that change the interaction of the phycobilisomes with the photosystem I and photosystem II core complexes. This mechanism is essential for cyanobacteria at low light intensities. Previous studies of cyanobacteria have identified a gene named rpaC, which appears to be specifically required for state transitions. The gene product of rpaC is very probably a transmembrane protein that is a structural component of the phycobilisome-photosystem II supercomplex. However, the physiological role of RpaC protein is unclear.Here we report the construction of an expression system that enables high production of fusion protein TrxHisTagSTag-RpaC, and describe suitable conditions for purification of this insoluble protein at a yield of 3 mg per 1 dm3 of bacterial culture. Cleavage with HRV 3C protease to remove the TrxHisTagSTag portion resulted in low yields of RpaC-protein (∼ 30 µg/dm3 of bacterial culture), therefore the applicability to structural studies was tested for the fusion protein only. Several preliminary conditions for crystallization of TrxHisTagSTag-RpaC were set up under which microcrystals were obtained. This set of conditions will be a good starting point for optimization in future crystallization experiments. TrxHisTagSTag-RpaC protein may prove useful in biochemical studies where the small size of RpaC protein is limiting the investigation of interactions with significantly larger parts of the photosynthetic apparatus. Furthermore, the purification procedure described here might also be applied to the production and purification of other small membrane proteins for biochemical and structural studies.

Reply to [ldquo]Structural requirements for RNA degradation by HIV-1 reverse transcriptase[rdquo]

In their correspondence, Das and colleagues 1 raise a number of issues about our work, which we address below. Firstly, we have not claimed that the RNA scissile phosphate is situated in the RNase H active site of HIV-1 RT ready for cleavage. Instead we reported 2 that our structures are “compatible with RNA degradation” (not “catalytically relevant” as incorrectly quoted by Das et al. 1), whereas all previous RT-nucleic acid (NA) complex structures are incompatible with RNA degradation. The incompatibility with RNA cleavage in the previous RT-NA complexes lies in the orientation of the NA substrate, not in the distance between NA and the RNase H active site, as we clearly demonstrated in Figure S5 2. In the previous RT-NA complexes, one DNA strand can be connected with the RNA/DNA hybrid positioned for RNA cleavage (Fig. 1a) according to the human and bacterial RNase H1-RNA/DNA hybrid structure 3,4, but the second strand (RNA equivalent) cannot be connected because of a 14 A gap. This gap cannot be closed by any amount of bending or unwinding of the duplex 3. In the three RT-RNA/DNA-NNRTI complex structures we reported 2, the RNA/DNA hybrids are oriented such that there is no long a gap (Fig. 1b), and a slight adjustment of the RNA strand would permit hydrolysis as illustrated in Figures 5 2. Indeed, the nearest RNA phosphate in our structures is 8.8 A from the active site carboxylate as depicted in Figure 5 2. We should clarify that the distance between the scissile phosphate and the active site in the human RNase H1-RNA/DNA complex is 4.6A (PDB: 2Q39) 3. Thus the scissile phosphate in the 4B3O structure needs to move 4.2A to be positioned for cleavage. It is not the distance but rather the NA orientation that makes our RNA/DNA hybrid compatible with RNA degradation, which is also evident in Fig. 1a of the correspondence by Das and colleague 1. Figure 1 Differences between previous RT-NA complexes and our RT-RNA/DNA structures. (a) All RT-NA structrues previously reported are similar 2 and represented here by the RT-DNA-dATP ternary complex (PDB:3KK2 6) in the polymerization mode. Human RNase H1-RNA/DNA structure ... Secondly, we disagree with the statement that our structure (4B3O) is most closely related to the RT-DNA/DNA-nevirapine structure (PDB 3V8I)5. Both the protein and nucleic acid of the 3V8I structure are more similar to that of RT-DNA complexes than to our RT-RNA/DNA hybrid complexes, as we showed in Figure 3 2. For example, the RNA/DNA hybrid in our structure has the A-form conformation, while the DNA duplex in 3V8I as well as all previously reported RT-NA complexes are largely B-form 2 (Fig. 1). Thirdly, with regard to the alleged crystal packing effects, we had noticed the lattice contact all along but found it irrelevant to RT-RNA/DNA hybrid complex formation, based on three different crystal forms of RT-RNA/DNA hybrid complexes (Figures 1 and S1 2). The region near the p66 thumb domain, with which Das and colleagues are concerned 1, is not involved in crystal packing in at least one of our three crystal structures 2, and the A-form conformation of our entire RNA/DNA is independent of this lattice contact. Fourthly, on the issue of the nick in the RNA strand used in one of the three RT-RNA/DNA complex structures presented in our paper, the nick was engineered by design as reported in the main text and Methods section, and depicted in Figures 1 and S1 2. However, the other two RT-RNA/DNA complex structures (PDB: 4B3P and 4B3Q) contain a continuous RNA strand 2. The statement by Das and colleagues that a continuous RNA/DNA duplex would not be able to adopt the conformation or trajectory adopted by the nicked hybrid 1 is simply incorrect. The RNA/DNA hybrids in all three of our structures, including two with a continuous RNA strand are in similar conformations (Figures 1d and S3 2). Finally, regarding cross-linking between RT and nucleic acid in previous RT complexes, we have not questioned the validity of this strategy in capturing HIV-1 RT in the DNA polymerization mode. Rather, we respectfully pointed out that in the more than 20 RT-DNA crosslinked structures, the DNAs are all in a similar conformation, and one that is incompatible with RNA degradation.volume 20 number 12 DeCember 2013 nature structural & molecular biology Kalyan Das1, Stefan G. Sarafianos2 & Eddy Arnold1 1Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey, USA. 2Christopher S. Bond Life Sciences Center, Department of Molecular Microbiology & Immunology and Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri, USA. e-mail: arnold@cabm.rutgers.edu