Simple SummaryAntibodies play a critical role in the immune system’s defense against pathogens. Such antibodies can be used for passive immunization, but they are rather expensive and difficult to produce. Alternatively, their structures can be used as a template for the design of peptides that retain the antibodies’ binding behaviour, but which are smaller in size and therefore more likely to be able to reach sterically shielded epitopes. However, identifying high-affinity sequences in antibody structures is challenging. In this context, we investigated the structural properties of the broadly neutralizing antibody PG16 that binds to the gp120 subunit of the HIV-1 envelope (Env) protein. Recognition occurs primarily through the complementarity determining region (CDR) H3 of PG16, which contains a tyrosine sulfation site. Molecular modeling and simulation of the protein dynamics reveal that the CDRH3 represents an energetic hotspot of PG16-gp120 recognition, and that sulfation plays an important role in enhancing the interaction. Moreover, we experimentally demonstrated that a CDRH3-derived peptide is still able to recognize gp120 with high affinity. Binding was further enhanced by the introduction of a disulfide bond that increases structural stability. These results suggest that the continued optimization of PG16-derived peptides might ultimately lead to an alternative therapeutic approach to combat HIV-1 infection.AbstractPG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by molecular dynamics simulations at the atomic level. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of modification and for the neighboring residues. This stabilization affects not only protein–protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Furthermore, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16, we obtained an experimental EC50value of 3nmfor the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Given their high affinity, it should be possible to further optimize the PG16-derived peptides as potential inhibitors of HIV invasion.