STRUCTURAL-CHANGES OF TUMOR-NECROSIS-FACTOR-ALPHA ASSOCIATED WITH MEMBRANE INSERTION AND CHANNEL FORMATION

Low pH enhances turner necrosis factor cu (TNF)-induced cytolysis of c ancer cells and TNF-membrane interactions that include binding, insert ion, and ion-channel formation, We have also found that TNF increases Na+ influx in cells, Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation, Fluor ometric studies link TNF's acid-enhanced membrane interactions to rapi d but reversible acquisition of hydrophobic surface properties, Intram embranous photolabeling shows that (i) protonation of TNF promotes mem brane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and i nsertion of TNF are two distinct events, Acidification relaxes the tri meric structure of soluble TNF so that the cryptic carboxyl termini, c entrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; ho wever, the proximal salt-bridged Lys-ll residues as well as regional s urface amino acids (Glu-23, Arg-32, and Arg-44) are notably more acces sible to proteases, The sequenced cleavage products bear the membrane- restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition, In summary, the trimer's structural plast icity is a major determinant of its channel-forming ability, Channel f ormation occurs when cracked or partially splayed trimers bind and pen etrate the bilayer, Reannealing leads to a slightly relaxed trimeric s tructure, The directionality of bilayer penetration conforms with x-ra y data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell mem brane.