What Does It Take to Bind CAR? Lane K. Law, Beverly L. Davidson  Molecular Therapy  Volume 12, Issue 4, Pages 599-609 (October 2005) DOI: 10.1016/j.ymthe.2005.05.017 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Schematic representations of the (A) Ad12, (B) Ad37, and (C) Ad30 knob monomers. These diagrams are adapted from Xia et al. [10] by permission of the publisher. Boxes represent individual amino acid residues. Numbers correspond to the position of each amino acid in the Ad12 knob. Residues found in loops are purple. Residues found in β strands are in blue. Loops and β strands are labeled, as well as the amino and carboxy termini. Critical CAR-binding residues (for Ad12) are green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 12, 599-609DOI: (10.1016/j.ymthe.2005.05.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Schematic representations of the (A) Ad12, (B) Ad37, and (C) Ad30 knob monomers. These diagrams are adapted from Xia et al. [10] by permission of the publisher. Boxes represent individual amino acid residues. Numbers correspond to the position of each amino acid in the Ad12 knob. Residues found in loops are purple. Residues found in β strands are in blue. Loops and β strands are labeled, as well as the amino and carboxy termini. Critical CAR-binding residues (for Ad12) are green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 12, 599-609DOI: (10.1016/j.ymthe.2005.05.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 The Ad12 knob–CAR-binding interface. (A) A view of the fiber trimer complexed with CAR. Knob monomers were yellow, blue, and gray in DeepView. The CAR D1 molecule was green. The AB loop of monomer A is red. The FG loop of the adjacent monomer B is pink. A white arrow indicates the interface between the Aad12knob and the CAR D1 molecule. (B) Key contact residues between the Ad12 AB loop and CAR D1. The AB loop of monomer A is red. The CAR D1 molecule is green. The side chains of Asp415, Pro417, Pro418, Leu426, and Lys429 of the Ad12 monomer A AB loop are visible, as are the side chains of Glu58, Val72, Leu75, Tyr82, Tyr85, Lys123, and Lys125 of the CAR D1 molecule. Carbon atoms are white. Oxygen atoms are red. Nitrogen atoms are blue. (C) Residues from the second monomer's FG loop (pink): Pro517, Pro519, Asn520, and Glu523. In particular Asn520 forms a hydrogen bond with Asp70 of CAR D1 (adapted from Bewley et al. [11] using DeepView, reproduced by permission of the publisher). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 12, 599-609DOI: (10.1016/j.ymthe.2005.05.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Primary amino acid alignment of the knob region of adenoviral serotypes 30, 37, 19, 15, 9, 2, 5, 12, 35, 3, 7, and 41L. Adenoviral serotypes 9, 2, 5, 12, and 41L can bind CAR (boxed in teal). Amino acids conserved among serotypes are shaded in gray. Residues critical for CAR binding have a green background. Residues boxed in brown are potentially inhibitory for CAR binding. Ad37 Lys240 (orange background) is required for binding to conjunctival cells. Ad30 sequences that may contribute to impaired CAR binding are indicated (orange boxes). The loops and strands are indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 12, 599-609DOI: (10.1016/j.ymthe.2005.05.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Primary amino acid alignment of the knob region of adenoviral serotypes 30, 37, 19, 15, 9, 2, 5, 12, 35, 3, 7, and 41L. Adenoviral serotypes 9, 2, 5, 12, and 41L can bind CAR (boxed in teal). Amino acids conserved among serotypes are shaded in gray. Residues critical for CAR binding have a green background. Residues boxed in brown are potentially inhibitory for CAR binding. Ad37 Lys240 (orange background) is required for binding to conjunctival cells. Ad30 sequences that may contribute to impaired CAR binding are indicated (orange boxes). The loops and strands are indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Molecular Therapy 2005 12, 599-609DOI: (10.1016/j.ymthe.2005.05.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Ad37 and Ad30 knob interactions with CAR. (A) Illustration of the bulge toward CAR present in the Ad37 FG loop. Amino acids Asp70 (CAR molecule) and Asn298 and Ser299 (Ad37 knob) are shown. A schematic diagram of the Ad37 monomer is depicted in Fig. 1B. A comparison of (A) with Fig. 2C illustrates the extent of the bulge. (B) Illustration of the bulge toward CAR present in the Ad30 FG loop. Amino acids Asp70 (CAR molecule) and Asp303 (Ad30 knob) are shown. Molecular Therapy 2005 12, 599-609DOI: (10.1016/j.ymthe.2005.05.017) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions