Cell, Volume 129

Supplemental Data

Structural Basis for the Inhibition of

Tyrosine Kinase Activity of ZAP-70 Sebastian Deindl, Theresa A. Kadlecek, Tomas Brdicka, Xiaoxian Cao, Arthur Weiss, and John Kuriyan

Table S1. Data Collection, Phasing, and Refinement Statistics

Data Collectiona

Space Group Unit Cell

# Molecules/asymmetric unit X-ray source Wavelength (Å) Resolution (Å) I/

Completeness (%) Rsym (%)

P1 a = 48.1 Å, b = 52.8 Å, c = 68.8 Å,

= 106.0°, = 93.1°, = 103.8° 1 Advanced Light Source, Berkeley, beamline 8.2.2 0.9537 50-2.6 16.2 (2.1) 95.3 (78.5) 8.7 (33.3)

Model Refinementb

Resolution (Å) # Reflections Rwork/Rfree

Rwork/Rfree (%) # Non-hydrogen protein/ligand atoms # Water molecules Rmsd bond length (Å) Rmsd angles (°) Average temperature (B) factor, protein (Å2)

50-2.6 15680/1622 22.0/29.0 4224/31 69 0.008 1.460 42.8

aValues as defined in SCALEPACK (Otwinowski and Minor, 1997); numbers in parentheses are statistics for the outer resolution shell (2.69-2.6 Å). bValues as defined in CNS (Brunger et al., 1998).

Figure S1. Conformers of the SH2-kinase linker.

Two alternate conformations for the SH2-kinase linker segment spanning residues 313-

322, as obtained from crystallographic simulated annealing twin refinements. The two

different conformers are colored green and blue.

Figure S2. Distance difference matrices.

Left: Structure of the kinase domain of ZAP-70 in the inactive Src/CDK-like

conformation. Regions that undergo structural changes upon adopting the active

conformation are colored. Right: Distance difference matrix between ZAP-70 in the

inactive Src/CDK-like conformation and ZAP-70 in the active conformation (PDB code:

1U59). Each entry in this symmetrical matrix represents the difference in the distance

between the corresponding C

atoms. C

atoms that come closer together on going from

the inactive Src/CDK-like conformation to the active conformation are indicated in

yellow/red. Distances that do not change much are green, and distances that expand are

blue. Data were cut off to +/- 5 Å.

Figure S3. Sequence alignment of ZAP-70 and Syk from different species, human

Src, human EphB2 and human Jak2.

Sequence alignment of ZAP-70/Syk with other tyrosine kinases. Secondary structure

elements (only for the kinase domain) are derived from the crystal structure of inactive

ZAP-70. Alignment was performed with ClustalW and formatted with ESPript using the

web interface.

Figure S4. Mutagenesis analysis of the interface.

LAT phosphorylation by diverse ZAP-70 mutants in the absence (left) or presence (right)

of Lck.

Figure S5.

The effect of hotspot mutations on ZAP-70 Y315F/Y319F dependent LAT

phosphorylation in the absence (A) or presence (B) of Lck.