Crystallography

Longer RE spacer inhibits p73 tetramer formation through biochemical and molecular mechanisms. X-ray crystallography showed that RE spacer length alters p73 DBD tertiary and quaternary structure, which in turn affects RE DNA conformation as well. As spacer length increases, dimer-dimer distance increases and tetramerization interface decreases.

Tetramerisation interface
As spacer lengthens, dimers rotate (14o in 4 bp spacer) and move away from each other. As a result, the tetramerisation interface decreases and even becomes absent in the 4-bp structure.

Dimer conformation
With such weaker tetramerisation restraints, dimerisation interfaces establish a distinct hydrogen-bond network, which alters conformation and increases stability of dimers, reducing their tetramerisation affinity.

The p73 tetramer conformations with different spacer lengths are shown in Figure 5a-d.

Figure 5a: 0-bp spacer (click for large image)

Figure 5b: 1-bp spacer (click for large image)

Figure 5c: 2-bp spacer (click for large image)

Figure 5d: 4-bp spacer (click for large image)

NMR spectroscopy showed that an additional helix in the C-terminal binding domain stabilizes p73 DBD tetramerisation. Mutations within it reduce van der Waals interaction between the two dimers. This suggests an additional determinant of p73 DBD tetramerisation.

DNA conformation

0-bp structure retains the ideal B form DNA. With 1-bp and 2-bp spacers, DNA unwinds into A form and slightly bends towards the major groove, in order to accommodate the extra bases in the same 3D space without disrupting tetramerisation. As tetramerisation is completely disrupted, 4-bp spacer rewinds into B form, showing a 3‎Å slide in the middle.

The DNA conformation determined by different spacer lengths are shown in Figure 6.

 

0-bp spacer                         1-bp spacer

 2-bp spacer                       4-bp spacer

Figure 6: Difference in p73 RE DNA conformation due to different spacer lengths