Currículo: Precisiones conceptuales

To understand the function of the DNA integrity scanning protein A (DisA) better, DisA from the thermophile bacterium Thermotoga maritima (TmaDisA) was crystallized by Gregor Witte and the atomic structure was solved (PDB entry 3C1Y). As shown in figure 9, TmaDisA crystallized in a long fiber-like chain of interconnected tetramers. The interface within the tetramer is especially mediated by the helical domain in the middle of the protein (shown in blue) and is with approximately 4400Å2 _{quite large and very likely a stable physiological interface. From size}

exclusion chromatography and analytical ultracentrifugation studies, it was already clear that DisA forms a complex in solution that is definitely larger than a tetramer. The results strongly support the existence of an octameric complex in solution.

MODEL 1

MODEL 2

Figure 9: Organization of DisA tetramers in the crystal. The two possible octameric DisA complexes are indicated. In model 1 (blue) two tetramers interact via the N-terminal nucleotide binding domains and in model 2 (orange) the interaction is mediated via the C-terminal DNA binding domains.

Figure 9 depicts the two different possible octameric assemblies, which can be found in the crystal structure: in model 1 the two tetramers interact via the N-terminal nucleotide binding domain (green) and in model 2 the interaction is mediated via the postulated DNA binding domain (orange) at the C-terminus. In model 1 the bound c-di-AMP ligand would be buried in the interface, whereas in model 2 it would be accessible to the solvent.

To identify which of the two octameric forms is existent in solution, SAXS studies were performed. SAXS curves were measured at different protein concentrations and no concentration dependent aggregation or the existence of attractive forces between the complexes could be observed. In addition, the sensitivity of the protein to radiation was analyzed by replicate exposures of the sample to X-rays and the complex seemed to be structurally very stable even after long exposure. The measured scattering curve of DisA is shown in figure 10A.

To determine the radius of gyration the ln(s) vs. s² - Guinier plot (Guinier, 1939) was used by analyzing the region of small s-values (see figure 10B). The Rg was determined from the slope of

the linear regression curve extrapolated to s² = 0 to be 52.8 Å for all protein concentrations (considering s · Rg < 1.3 to fulfill the constraints for the Guinier approximation).

For transformation of the scattering data into the pair-distribution function, the maximum particle diameter Dmax was determined to be 176 Å, which fits very well to the longest distance of

slightly less than 175 Å in the crystal structure. The calculated p(r)-distribution is shown in figure 10C and shows the typical shape of an elongated, cylindrical molecule.

A

B

C

Figure 10: SAXS studies with DisA from T. maritima. A: Scattering curve of DisA. The buffer is already sub- tracted and the intensity is plotted logarithmically. B: Linear region of the Guinier plot, from which Rg and I0

can be derived. C: Pair distribution function of DisA, which was used for

ab inito modeling with GASBORp.

The solution structure was determined using the p(r) distribution and the dummy residue approach (Svergun et al., 2001) with different symmetry constraints for modeling. As the DisA structure shows a 4-fold symmetry and a 2-fold symmetry with perpendicular symmetry axes, four different solution structures were calculated: implicating no symmetry (P1), one forced 2- fold symmetry axis (P2), one forced 4-fold symmetry axis (P4) and forced 2- and 4-fold symmetry axis (P42). For all four structures the ab initio structure calculation was repeated 10

1 10 100 1000 10000 0 0.1 0.2 0.3 s [1/Å] lg I (s ) 7.5 8 8.5 9 9.5 0 0.0004 0.0008 s²[1/Ų] ln I (s ) 0 2 4 6 8 0 50 100 150 r[Å] p (r ), r e la ti ve

times using identical input parameters and the structures were then aligned and averaged. The obtained structures are shown in figure 11 as bead models.

Figure 11: DisA solution structures calculated with different implicated symmetries. The depicted bead models of SAXS solution structures were calculated with GASBORp using the pair distribution function of DisA shown in figure 9C. 10 structures were aligned and averaged. For ab initio modeling different symmetry constraints were used: no symmetry (orange), 2-fold (green) and 4-fold (blue) and both 2- and 4-fold symmetry (yellow).

The final bead model calculated with forced single 4-fold symmetry did not give a reasonable result, because the program has located the symmetry axis in a wrong orientation. All other three structures show a similar overall shape.

To determine which of the two possible crystallographic octamers is present in solution, the crystal structures were overlaid with the ab initio solution structure using the SUPCOMB program (Kozin and Svergun, 2000). The superposition of the bead model obtained with the 2- fold symmetry with both octamer models is shown in figure 12. Model 1 with facing N-termini of DisA fits very well to the solution structure, whereas for model 2 some structural elements clearly stick out of the envelope and are missing at other positions.

In addition to comparison at structural level, the two models were compared with the SAXS data at the level of the scattering curves.

The theoretical scattering curves of both possible octameric models were calculated using CRYSOL and overlaid with the measured scattering curve of the DisA complex. Figure 13 shows the comparison between calculated and measured scattering data. Whereas the calculated

scattering curve for model 1 only shows small deviations from the measured data, model 2 clearly does not fit the experimental scattering curve. Thus, model 1 is existent in solution and the crystal structure of DisA resembles its solution structure very much.

Figure 12: Superposition of the two possible octamer models with the SAXS structure. Clearly model 1 (top) fits better to the solution structure than model 2.

0 2 4 0 0.05 0.1 0.15 0.2 0.25 s [1/Å] lg I (s )

Figure 13: Experimental scattering curve in comparison to theoretical scattering curves calculated from the two possible DisA assemblies: The calculated scattering curve for model 1 (solid blue line) agrees very well with the experimental curve (black crosses), while the calculated curve for model 2 (dashed orange line) substantially disagrees.