Summary How the epidermal growth factor receptor (EGFR) activates is incompletely understood. The intracellular portion of the receptor is intrinsically active in solution, and to study its regulation we measured autophosphorylation as a function of EGFR surface density in cells. Without EGF, intact EGFR escapes inhibition only at high surface densities. While the transmembrane helix and the intracellular module together suffice for constitutive activity even at low densities, the intracellular module is inactivated when tethered on its own to the plasma membrane and fluorescence cross-correlation shows that it fails to dimerize. NMR and functional data indicate that activation requires an N-terminal interaction between the transmembrane helices, which promotes an antiparallel interaction between juxtamembrane segments and release of inhibition by the membrane. We conclude that EGF binding removes steric constraints in the extracellular module, promoting activation through N-terminal association of the transmembrane helices.

Two-dimensional NMR has been used to study the 2:1 distamycin A-d(CGCAAATTGGC)-d(GCCAA-TTTGCG) complex. The nuclear Overhauser effect spectroscopy (NOESY) experiment was used to assign the aromatic and C1'H DNA protons and to identify drug-DNA contacts. These data indicate that two drug molecules bind simultaneously in the minor groove of the central 5'-AAATT-3' segment and are in close contact with both the DNA and one another. One drug binds with the formyl end close to the second adenine base of the A-rich strand, while the other drug binds with the formyl end close to the second adenine of the complementary strand. With this binding orientation, the positively charged propylamidinium groups are directed toward opposite ends of the helix. Molecular modeling shows that the minor groove must expand relative to the 1:1 complex to accommodate both drugs. Energy calculations suggest that electrostatic interactions, hydrogen bonds, and van der Waals forces contribute to the stability of the complex.An understanding of drug-DNA interactions at the molecular level is important in facilitating the design of new drugs and probes that can recognize specific DNA sequences. Distamycin A is an oligopeptide antibiotic ( Fig. 1) that binds preferentially to the minor groove of A+T-rich DNA sites (1, 2). Recently, several studies of 1:1 distamycin A-DNA complexes have provided insight into both the specificity and the forces responsible for the tight binding of this drug. Footprinting and affinity cleaving studies (3) have shown that distamycin A binds tightly to 5'-AAATT-3' in two orientations to a degree dependent on flanking base pairs and, in general, prefers sites that contain several adjacent adenine residues. NMR studies of distamycin A with d(CGCGAAT-TCGCG)2 (4, 5) have shown that 5'-AATT-3' is a good binding site, while calorimetric studies of distamycin A with d(GCGAATTCGC)2 (6) indicate that the binding constant for the complex is near 3 x 108 M-1, and that the binding process is enthalpy driven. Recent crystallographic studies of distamycin A with d(CGCAAA1TJ7GCG)2 (7) revealed that the drug bound to a 5'-ATTT-3' sequence, although other sites with four A-T base pairs were available. Similar results have been obtained from footprinting (8, 9), calorimetric (10), crystallographic (11)(12)(13)(14), and NMR (15-18) studies of netropsin-DNA complexes. Analysis of these data indicates that van der Waals forces, hydrogen bonds, and electrostatic forces (19) contribute to the stability of these complexes and the sequence specificity of these drugs.Using than the minimal four A-T base pairs was available. Titration of the DNA with distamycin A showed that at low drugto-DNA ratios at least two different binding sites were occupied. The binding behavior in this regime will be described elsewhere. At higher amounts of added drug, but still below a 1:1 ratio, a new binding mode was evident; a complex using this mode became the only form present at a drugto-DNA ratio of 2:1. It is the structure of this 2:1 complex whic...

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