Structure of Mycobacterium tuberculosis single-stranded DNA-binding protein. Variability in quaternary structure and its implications

Single-stranded DNA-binding protein (SSB) is an essential protein necessary for the functioning of the DNA replication, repair and recombination machineries. Here we report the structure of the DNA-binding domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different crystals distributed in two forms. The structure of one of the forms was solved by a combination of isomorphous replacement and anomalous scattering. This structure was used to determine the structure of the other form by molecular replacement. The polypeptide chain in the structure exhibits the oligonucleotide binding fold. The globular core of the molecule in different subunits in the two forms and those in Escherichia coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar structure, although the three loops exhibit considerable structural variation. However, the tetrameric MtuSSB has an as yet unobserved quaternary association. This quaternary structure with a unique dimeric interface lends the oligomeric protein greater stability, which may be of significance to the functioning of the protein under conditions of stress. Also, as a result of the variation in the quaternary structure the path adopted by the DNA to wrap around MtuSSB is expected to be different from that of EcoSSB.     

The congenital bicuspid aortic valve can experience high-frequency unsteady shear stresses on its leaflet surface

The bicuspid aortic valve (BAV) is a common congenital malformation of the aortic valve (AV) affecting 1% to 2% of the population. The BAV is predisposed to early degenerative calcification of valve leaflets, and BAV patients constitute 50% of AV stenosis patients. Although evidence shows that genetic defects can play a role in calcification of the BAV leaflets, we hypothesize that drastic changes in the mechanical environment of the BAV elicit pathological responses from the valve and might be concurrently responsible for early calcification. An in vitro model of the BAV was constructed by surgically manipulating a native trileaflet porcine AV. The BAV valve model and a trileaflet AV (TAV) model were tested in an in vitro pulsatile flow loop mimicking physiological hemodynamics. Laser Doppler velocimetry was used to make measurements of fluid shear stresses on the leaflet of the valve models using previously established methodologies. Furthermore, particle image velocimetry was used to visualize the flow fields downstream of the valves and in the sinuses. In the BAV model, flow near the leaflets and fluid shear stresses on the leaflets were much more unsteady than for the TAV model, most likely due to the moderate stenosis in the BAV and the skewed forward flow jet that collided with the aorta wall. This additional unsteadiness occurred during mid- to late-systole and was composed of cycle-to-cycle magnitude variability as well as high-frequency fluctuations about the mean shear stress. It has been demonstrated that the BAV geometry can lead to unsteady shear stresses under physiological flow and pressure conditions. Such altered shear stresses could play a role in accelerated calcification in BAVs.     

Insights into Chi recognition from the structure of an AddAB-type helicase-nuclease complex

In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is dependent upon the recombination hotspot sequence Chi and is catalysed by either an AddAB- or RecBCD-type helicase-nuclease. Here, we report the crystal structure of AddAB bound to DNA. The structure allows identification of a putative Chi-recognition site in an inactivated helicase domain of the AddB subunit. By generating mutant protein complexes that do not respond to Chi, we show that residues responsible for Chi recognition are located in positions equivalent to the signature motifs of a conventional helicase. Comparison with the related RecBCD complex, which recognizes a different Chi sequence, provides further insight into the structural basis for sequence-specific ssDNA recognition. The structure suggests a simple mechanism for DNA break processing, explains how AddAB and RecBCD can accomplish the same overall reaction with different sets of functional modules and reveals details of the role of an Fe-S cluster in protein stability and DNA binding.     

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp.     

In-vivo mitral annuloplasty ring transducer: Implications for implantation and annular downsizing

Mitral annuloplasty has been a keystone to the success of mitral valve repair in functional mitral regurgitation. Understanding the complex interplay between annular-ring stresses and left ventricular function has significant implications for patient-ring selection, repair failure, and patient safety. A step towards assessing these challenges is developing a transducer that can be implanted in the exact method as commercially available rings and can quantify multidirectional ring loading. An annuloplasty ring transducer was developed to measure stresses at eight locations on both the in-plane and out-of-plane surfaces of an annuloplasty ring's titanium core. The transducer was implanted in an ovine subject using 10 sutures at near symmetric locations. At implantation, the ring was observed to undersize the mitral annulus. The flaccid annulus exerted both compressive (−) and tensile stresses (+) on the ring ranging from −3.17 to 5.34 MPa. At baseline hemodynamics, stresses cyclically changed and peaked near mid-systole. Mean changes in cyclic stress from ventricular diastole to mid-systole ranged from −0.61 to 0.46 MPa (in-plane direction) and from −0.49 to 1.13 MPa (out-of-plane direction). Results demonstrate the variability in ring stresses that can be introduced during implantation and the cyclic contraction of the mitral annulus. Ring stresses at implantation were approximately 4 magnitudes larger than the cyclic changes in stress throughout the cardiac cycle. These methods will be extended to ring transducers of differing size and geometry. Upon additional investigation, these data will contribute to improved knowledge of annulus-ring stresses, LV function, and the safer development of mitral repair techniques.     

DNA binding to RecD: role of the 1B domain in SF1B helicase activity

The molecular mechanism of superfamily 1Balpha helicases remains unclear. We present here the crystal structure of the RecD2 helicase from Deinococcus radiodurans at 2.2-A resolution. The structure reveals the folds of the 1B and 2B domains of RecD that were poorly ordered in the structure of the Escherichia coli RecBCD enzyme complex reported previously. The 2B domain adopts an SH3 fold which, although common in eukaryotes, is extremely rare in bacterial systems. In addition, the D. radiodurans RecD2 structure has aided us in deciphering lower resolution (3.6 A) electron density maps for the E. coli RecBCD enzyme in complex with a long DNA substrate that interacts with the RecD subunit. Taken together, these structures indicated an important role for the 1B domain of RecD, a beta-hairpin that extends from the surface of the 1A domain and interacts with the DNA substrate. On the basis of these structural data, we designed a mutant RecD2 helicase that lacks this pin. The 'pin-less' mutant protein is a fully active ssDNA-dependent ATPase but totally lacks helicase activity.     

Isolated effect of geometry on mitral valve function for in silico model development

Computational models for the heart's mitral valve (MV) exhibit several uncertainties that may be reduced by further developing these models using ground-truth data-sets. This study generated a ground-truth data-set by quantifying the effects of isolated mitral annular flattening, symmetric annular dilatation, symmetric papillary muscle (PM) displacement and asymmetric PM displacement on leaflet coaptation, mitral regurgitation (MR) and anterior leaflet strain. MVs were mounted in an in vitro left heart simulator and tested under pulsatile haemodynamics. Mitral leaflet coaptation length, coaptation depth, tenting area, MR volume, MR jet direction and anterior leaflet strain in the radial and circumferential directions were successfully quantified at increasing levels of geometric distortion. From these data, increase in the levels of isolated PM displacement resulted in the greatest mean change in coaptation depth (70% increase), tenting area (150% increase) and radial leaflet strain (37% increase) while annular dilatation resulted in the largest mean change in coaptation length (50% decrease) and regurgitation volume (134% increase). Regurgitant jets were centrally located for symmetric annular dilatation and symmetric PM displacement. Asymmetric PM displacement resulted in asymmetrically directed jets. Peak changes in anterior leaflet strain in the circumferential direction were smaller and exhibited non-significant differences across the tested conditions. When used together, this ground-truth data-set may be used to parametrically evaluate and develop modelling assumptions for both the MV leaflets and subvalvular apparatus. This novel data may improve MV computational models and provide a platform for the development of future surgical planning tools.     

Substitutions at tyrosine 66 of Escherichia coli uracil DNA glycosylase lead to characterization of an efficient enzyme that is recalcitrant to product inhibition

Uracil DNA glycosylase (UDG), a ubiquitous and highly specific enzyme, commences the uracil excision repair pathway. Structural studies have shown that the tyrosine in a highly conserved GQDPY water-activating loop of UDGs blocks the entry of thymine or purines into the active site pocket. To further understand the role of this tyrosine (Y66 in Escherichia coli UDG), we have overproduced and characterized Y66F, Y66H, Y66L and Y66W mutants. The complexes of the wild-type, Y66F, Y66H and Y66L UDGs with uracil DNA glycosylase inhibitor (Ugi) (a proteinaceous substrate mimic) were stable to 8 M urea. However, some dissociation of the complex involving the Y66W UDG occurred at this concentration of urea. The catalytic efficiencies (V_max / K_m) of the Y66L and Y66F mutants were similar to those of the wild-type UDG. However, the Y66W and Y66H mutants were ~7- and ~173-fold compromised, respectively, in their activities. Interestingly, the Y66W mutation has resulted in an enzyme which is resistant to product inhibition. Preferential utilization of a substrate enabling a long range contact between the –5 phosphate (upstream to the scissile uracil) and the enzyme, and the results of modeling studies showing that the uracil-binding cavity of Y66W is wider than those of the wild type and other mutant UDGs, suggest a weaker interaction between uracil and the Y66W mutant. Furthermore, the fluorescence spectroscopy of UDGs and their complexes with Ugi, in the presence of uracil or its analog, 5-bromouracil, suggests compromised binding of uracil in the active site pocket of the Y66W mutant. Lack of inhibition of the Y66W UDG by apyrimidinic DNA (AP-DNA) is discussed to highlight a potential additional role of Y66 in shielding the toxic effects of AP-DNA, by lowering the rate of its release for subsequent recognition by an AP endonuclease.     

Experimental measurement of dynamic fluid shear stress on the aortic surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. Although exact causes and mechanisms of AV calcification are unclear, previous studies suggest that mechanical forces play a role. Since calcium deposits occur almost exclusively on the aortic surfaces of AV leaflets, it has been hypothesized that adverse patterns of fluid shear stress on the aortic surface of AV leaflets promote calcification. The current study characterizes AV leaflet aortic surface fluid shear stresses using Laser Doppler velocimetry and an in vitro pulsatile flow loop. The valve model used was a native porcine valve mounted on a suturing ring and preserved using 0.15% glutaraldehyde solution. This valve model was inserted in a mounting chamber with sinus geometries, which is made of clear acrylic to provide optical access for measurements. To understand the effects of hemodynamics on fluid shear stress, shear stress was measured across a range of conditions: varying stroke volumes at the same heart rate and varying heart rates at the same stroke volume. Systolic shear stress magnitude was found to be much higher than diastolic shear stress magnitude due to the stronger flow in the sinuses during systole, reaching up to 20 dyn/cm² at mid-systole. Upon increasing stroke volume, fluid shear stresses increased due to stronger sinus fluid motion. Upon increasing heart rate, fluid shear stresses decreased due to reduced systolic duration that restricted the formation of strong sinus flow. Significant changes in the shear stress waveform were observed at 90 beats/min, most likely due to altered leaflet dynamics at this higher heart rate. Overall, this study represents the most well-resolved shear stress measurements to date across a range of conditions on the aortic side of the AV. The data presented can be used for further investigation to understand AV biological response to shear stresses.     

X-ray structural studies of Mycobacterium tuberculosis RRF and a comparative study of RRFs of known structure. Molecular plasticity and biological implications

The crystal structure of Mycobacterium tuberculosis ribosome recycling factor has been determined and refined against three X-ray diffraction data sets, two collected at room temperature and the other at 100K. The two room-temperature data sets differ in the radiation damage suffered by the crystals before the data used for processing were collected. A comparison between the structures refined against the two data sets indicates the possibility of radiation-induced conformational change. The L-shaped molecule is composed of a long three-helix bundle domain (domain I) and a globular domain (domain II) connected by a linker region. The main difference between the room-temperature structure and the low temperature structure is in the rotation of domain II about an axis close to its libration axis. This observation and a detailed comparative study of ribosome recycling factors (RRFs) of known structures led to an elaboration of the present understanding of the structural variability of RRF. The variability involves a change in the angle between the two arms of the molecule, a rotation of domain II in a plane nearly perpendicular to the axis of the helix bundle and an internal rotation of domain II. Furthermore, the domains and the linker could be delineated into fixed and variable regions in a physically meaningful manner. The relative mobility of the domains of the molecule in the crystal structure appears to be similar to that in the ribosome--RRF complex. That permits a meaningful discussion of the structural features of RRF in terms of ribosome--RRF interactions. The structure also provides insights into the results of inter-species complementation studies.