The
rmodynamic measu
rements, st
ructu
ral dete
rminations,and molecula
r computations we
reapplied to a se
ries of peptide ligands of the pp60
c-srcSH2 domain in an attempt to unde
rstand the c
riticalbinding dete
rminants fo
r this class of molecules. Isothe
rmaltit
ration calo
rimet
ry (ITC) measu
rementswe
re combined with st
ructu
ral data de
rived f
rom X-
ray c
rystallog
raphicstudies on 12 peptide-SH2 domaincomplexes. The peptide ligands studied fall into two gene
ralclasses: (1) dipeptides of the gene
ralf
ramewo
rk
N-acetylphosphoty
rosine (o
r phosphoty
rosine
replacement)-Glu o
r methionine (o
r S-methylcysteine)-X, whe
re X
rep
resents a hyd
rophobic amine, and (2) tet
ra- o
rpentapeptides of the gene
ralf
ramewo
rk
N-acetylphosphoty
rosine-Glu-Glu-Ile-X, whe
re X
rep
resents eithe
r Glu, Gln, o
r NH
2.Dipeptideanalogs which featu
red X as eithe
r hexanolamine o
r heptanolamine we
reable to pick up new hyd
rogenbonds involving thei
r hyd
roxyl g
roups within a p
redominantly lipophilicsu
rface cavity. Howeve
r, dueto inte
rnal st
rain as well as the solvent accessibility of the newhyd
rogen bonds fo
rmed, no net inc
reasein binding affinity was obse
rved. Phosphatase-
resistantbenzylmalonate and
![](/images/gifcha<font color=)
rs/alpha.gif" BORDER=0>,
![](/images/gifcha<font color=)
rs/alpha.gif" BORDER=0>-difluo
robenzyl phosphonate analogs of phosphoty
rosine
retained some binding affinity fo
rthe pp60
c-src SH2 domain butcaused local st
ructu
ral pe
rtu
rbations in the phosphoty
rosine-bindingsite. In the case whe
re a
reve
rsiblecovalent thiohemiacetal was fo
rmed between a fo
rmylated phosphoty
rosineanalog and the thiol side chainof Cys-188,
![](/images/gifcha<font color=)
rs/Delta.gif" BORDER=0 >
S was 25.6 cal/(mol K) lowe
r than fo
r thenonfo
rmylated phosphoty
rosine pa
rent. No
rmalmode calculations show that the d
ramatic dec
rease in ent
ropy obse
rvedfo
r the covalent thiohemiacetalcomplex is due to the inability of the phosphoty
rosine moiety tot
ransfo
rm lost
rotational and t
ranslationaldeg
rees of f
reedom into new vib
rational modes.