Caracterización y análisis de ideas previas (Prueba de entrada)

Candida glabrata

The variant Sc118+129 of the SH2 domain showed an optimal behavior in terms of solubility and in limited proteolysis experiments (2.2.6, data not shown) but did not form crystals (3.3.1). Due to the high conservation of the domain in various species, the domain borders that were delineated in S. cerevisiae could be transferred to the proteins of Candida glabrata, Schizosaccharomyces pombe and Homo sapiens (Fig. 9 A). In the new variants two additional residues were removed from the C-terminus, based on sequence alignment and secondary structure prediction. The coding sequences for these domain variants were cloned, expressed and purified (chapters 2.2.1, 2.2.3, 2.2.5 and 3.2.2). These variants showed a similar behaviour in the purification compared to the S. cerevisiae protein and were purified to high homogeneity. The quality of the protein sample is exemplified for the

C. glabrata variant in Fig 7 A, but similar results were obtained for the domains from the other organisms.

Initial screenings for formation of crystals gave positive results only for the C. glabrata

protein. Refinement of these conditions, including streak seeding, lead to an overall improvement of crystals and to a native dataset to 2.8 Å resolution (see 3.2.4 and Fig 7 A). However, despite several attempts to solve the structure by molecular replacement with different search models (not shown), no solution could be obtained.

Thus we attempted to get de novo phases from anomalous diffraction of Selenium atoms, by the incorporation of seleno-methionine (2.2.3). For this we had to insert several point mutations into the sequence of the C. glabrata SH2 domain, since the native protein did not contain any methionine residue. 5 point mutations were designed (see 3.2.3 and Fig. 5). Four of those could be cloned (mutations A, C,D and E) and the resulting proteins were soluble. Mutations C, D and E were chosen for further subcloning, resulting in double mutant CD and triple mutant CDE, which again showed high solubility. A repeated screening for inital crystallization conditions with the two mutant proteins revealed two novel crystallization conditions for mutant CD (buffers Spt6 SH2-1 and -2, chapter 2.1.4, Table 13). Mutant CDE was crystallized in similar conditions as the native protein, but a SAD dataset to 3.1 Å resolution did not result in solution of the structure because of a weak anomalous signal (data not shown). However, crystals from mutant CD were suitable to solve the structure of the SH2 domain in multiple wavelength anomalous diffraction experiment (MAD, 3.2.5 , Table 16). Subsequently, also the second crystal form of the CD mutant could be solved by molecular replacement, using the structure from crystal form 1 as a search model (Table 16). All diffraction quality crystals that were obtained are summarized in Fig. 7 B.

3 Structure and requirement of the Spt6 SH2 domain 42

A

B

Figure 7: Quality of the C. glabrata SH2 domain protein sample and the resulting diffraction quality crystals

(A) Chromatogram of a Superose12 size exclusion chromatography of the C. glabrata Spt6 SH2 domain. Absorption units at 280 nm and 260 nm are shown in blue and red, respectively. In addition, an overloaded, Coomassie stained SDS-PAGE of the pooled peak fractions is shown, to demonstrate the purity of the protein sample.

(B) In columns, the different crystals of the C. glabrata Spt6 SH2 domain, the respective crystallization buffers (see also 2.1.4, Table 13), diffraction images and resolution, experiment and results are shown.

3 Structure and requirement of the Spt6 SH2 domain 43 Table 16: X-ray diffraction and refinement statistics for C. glabrata Spt6 selenomethionine double mutant CD crystals

3.3.3 Crystallization of SH2 domains from various species

Interestingly, the SH2 domains of S. cerevisiae and C. glabrata only differ in 10 amino acid positions, as is shown in the alignment left in Fig. 8. Although most of these positions are highly conserved, they make the difference between crystal formation or no crystals. When these residues are mapped on the four molecules of the asymmetric unit of the CD mutant crystal form 1, all residues except for two (A1291 and V1304) lie on the surface of the individual molecules and thus in between the molecules that build up the crystals (Fig. 8, right). These „evolutionary point mutations“ render the C. glabrata protein variant suitable for crystallization. Thus, making use of naturally occurring variances in proteins by extending crystallization trials to different source organisms is an appropriate remedy in the crystallization of difficult proteins.

crystal form 1 crystal form 2

Data collection

Space group P65 P32

Cell dimensions

a, b, c (Å) 54.5, 54.5, 253.4 71.6, 71.6, 87.6

, , (°) 90, 90, 120 90, 90, 120

Peak Remote Inflection

Wavelength (Å) 0.97973 0.90810 0.97987 0.97971 Resolution (Å) 20-1.9 20-1.9 20-1.9 20-2.4 Rsym (%) 5.2 (12.9) 5.3 (22.3) 3.9 (12.6) 6.2 (19.0) I / I 42.9 (7.3) 34.35 (7.5) 31.24 (6.0) 45.14 (7.7) Completeness (%) 99.4 (96.6) 99.9 (100) 99.3 (95.7) 99.5 (95.4) Redundancy 4.2 (2.8) 7.7 (6.7) 4.1 (2.8) 3.9 (3.7) Refinement Resolution (Å) 1.9 2.4 No. reflections 33162 18513 Rwork / Rfree (%) 19.6 / 24.1 25.27 / 28.50 No. atoms Protein 3292 3160 Ligand/ion 20 - Water 428 76 B-factors Protein 26.6 29.8 Ligand/ions 23.5 - Water 35.4 27.8 R.m.s deviations Bond lengths (Å) 0.005 0.007 Bond angles (°) 1.2 1.4

3 Structure and requirement of the Spt6 SH2 domain 44

Figure 8: The crystallizable C. glabrata protein variant differs in only 10 amino acid positions from the

S. cerevisiae protein

Alignment of the S. cerevisiae and C.glabrata SH2 domain sequences (left). Invariant residues are coloured in green, conserved residues are indicated in orange (high) and yellow (low), unconserved residues in gray. On the right, the 4 molecules of the asymmetric unit of crystal „SeMet mutant CD“ is shown. Invariant residues from the alignment are shown as a ribbon model (different greens for every molecule in the asymmetric unit). Conserved and unconserved residues are shown as stick models with the same colours as in the alignment.

3.3.4 The structure of the Spt6 SH2 domain reveals a typical SH2 fold