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Lectins, carbohydrate-binding proteins, have been regarded as potential antiviral agents, as some can bind glycans on viral surface glycoproteins and inactivate their functions. However, clinical development of lectins has been stalled by the mitogenicity of many of these proteins, which is the ability to stimulate deleterious proliferation, especially of immune cells. We previously demonstrated that the mitogenic and antiviral activities of a lectin (banana lectin, BanLec) can be separated via a single amino acid mutation, histidine to threonine at position 84 (H84T), within the third Greek key. The resulting lectin, H84T BanLec, is virtually non-mitogenic but retains antiviral activity. Decreased mitogenicity was associated with disruption of pi–pi stacking between two aromatic amino acids. To examine whether we could provide further proof-of-principle of the ability to separate these two distinct lectin functions, we identified another lectin, Malaysian banana lectin (Malay BanLec), with similar structural features as BanLec, including pi–pi stacking, but with only 63% amino acid identity, and showed that it is both mitogenic and potently antiviral. We then engineered an F84T mutation expected to disrupt pi–pi stacking, analogous to H84T. As predicted, F84T Malay BanLec (F84T) was less mitogenic than wild type. However, F84T maintained strong antiviral activity and inhibited replication of HIV, Ebola, and other viruses. The F84T mutation disrupted pi–pi stacking without disrupting the overall lectin structure. These findings show that pi–pi stacking in the third Greek key is a conserved mitogenic motif in these two jacalin-related lectins BanLec and Malay BanLec, and further highlight the potential to rationally engineer antiviral lectins for therapeutic purposes. [ABSTRACT FROM AUTHOR]

A challenging objective of de novo metalloprotein design is to control of the outer coordination spheres of an active site to fine tune metal properties. The well‐defined three stranded coiled coils, TRI and CoilSer peptides, are used to address this question. Substitution of Cys for Leu yields a thiophilic site within the core. Metals such as HgII, PbII, and AsIII result in trigonal planar or trigonal pyramidal geometries; however, spectroscopic studies have shown that CdII forms three‐, four‐ or five‐coordinate CdIIS3(OH2)x (in which x=0–2) when the outer coordination spheres are perturbed. Unfortunately, there has been little crystallographic examination of these proteins to explain the observations. Here, the high‐resolution X‐ray structures of apo‐ and mercurated proteins are compared to explain the modifications that lead to metal coordination number and geometry variation. It reveals that Ala substitution for Leu opens a cavity above the Cys site allowing for water excess, facilitating CdIIS3(OH2). Replacement of Cys by Pen restricts thiol rotation, causing a shift in the metal‐binding plane, which displaces water, forming CdIIS3. Residue d‐Leu, above the Cys site, reorients the side chain towards the Cys layer, diminishing the space for water accommodation yielding CdIIS3, whereas d‐Leu below opens more space, allowing for equal CdIIS3(OH2) and CdIIS3(OH2)2. These studies provide insights into how to control desired metal geometries in metalloproteins by using coded and non‐coded amino acids. [ABSTRACT FROM AUTHOR]

Although metal ion binding to naturally occurring l-amino acid proteins is well documented, understanding the impact of the opposite chirality ( d-)amino acids on the structure and stereochemistry of metals is in its infancy. We examine the effect of a d-configuration cysteine within a designed l-amino acid three-stranded coiled coil in order to enforce a precise coordination number on a metal center. The d chirality does not alter the native fold, but the side-chain re-orientation modifies the sterics of the metal binding pocket. l-Cys side chains within the coiled-coil structure have previously been shown to rotate substantially from their preferred positions in the apo structure to create a binding site for a tetra-coordinate metal ion. However, here we show by X-ray crystallography that d-Cys side chains are preorganized within a suitable geometry to bind such a ligand. This is confirmed by comparison of the structure of ZnIICl(CSL16DC)32− to the published structure of ZnII(H2O)(GRAND-CSL12AL16LC)3−. Moreover, spectroscopic analysis indicates that the CdII geometry observed by using l-Cys ligands (a mixture of three- and four-coordinate CdII) is altered to a single four-coordinate species when d-Cys is present. This work opens a new avenue for the control of the metal site environment in man-made proteins, by simply altering the binding ligand with its mirror-imaged d configuration. Thus, the use of non-coded amino acids in the coordination sphere of a metal promises to be a powerful tool for controlling the properties of future metalloproteins. [ABSTRACT FROM AUTHOR]