PDBsum entry 3csg

De novo protein, sugar binding protein

PDB id: 3csg

Contents

Protein chain

458 a.a.

Waters ×284

References listed in PDB file

Key reference

Title

A dominant conformational role for amino acid diversity in minimalist protein-Protein interfaces.

Authors

R.N.Gilbreth, K.Esaki, A.Koide, S.S.Sidhu, S.Koide.

Ref.

J Mol Biol, 2008, 381, 407-418. [DOI no: 10.1016/j.jmb.2008.06.014]

PubMed id

18602117

Abstract

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed "monobodies." One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.

Figure 1.
Fig. 1. The FNfn10 scaffold and loop sequences of MBP-binding monobodies. (a) A schematic of the FNIII scaffold. The seven β-strands are labeled A–G and three loops used for interface design (BC, DE and FG) are labeled. (b) The amino acid sequences of the loop regions of YS-only monobody YS1 and monobodies from the YSX library. For the latter group, the number of times each sequence was recovered is indicated in parentheses. The dissociation constant for MBP as measured by yeast display is also given. Unmutated residues originating from the mutagenesis template are colored gray.

Figure 4.
Fig. 4. The paratope structures of the YS1 and YSX1 monobodies. (a) The surface area buried at the interface of individual residues in the BC and FG loops of YS1 and YSX1. (b) The interface buried surface area contributed by each amino type to the YS1 and YSX1 paratopes. (c and d) Structural details of the YS1 and YSX1 interfaces. The left panels show an overall view of the arrangement of major interface contacts and the right panels show close-up views of these interactions. In these illustrations, MBP is shown as a gray surface/sticks and contacting monobody paratope residues are shown as sticks. The carbon atoms of the FG loop residues are colored green, and those of the BC loop residues cyan. Putative hydrogen bonds are indicated by dashed lines.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2008, 381, 407-418) copyright 2008.

Secondary reference #1

Title

High-Affinity single-Domain binding proteins with a binary-Code interface.

Authors

A.Koide, R.N.Gilbreth, K.Esaki, V.Tereshko, S.Koide.

Ref.

Proc Natl Acad Sci U S A, 2007, 104, 6632-6637. [DOI no: 10.1073/pnas.0700149104]

PubMed id

17420456

Figure 1.
Fig. 1. Amino acid sequences of Y/S monobodies. (A) A schematic drawing of the monobody scaffold. -Strands A–G and the three loops that are diversified in the library are indicated. (B) Affinity and amino acid sequences of Y/S monobodies that were selected from the initial library selection. The number of occurrences for clones that appeared more than once is indicated in parentheses. K[d] values determined by using yeast surface display are also shown. The sequences for the three loops are shown, with the numbering of Main et al. (20). Tyr, Ser, and the other amino acids are shaded in yellow, red, and gray, respectively.

Figure 4.
Fig. 4. The binding interface of the MBP-74 monobody and MBP. (A) The monobody paratope residues are shown as stick models, and the MBP epitope surface is shown in the same manner as in Fig. 3D. The carbon atoms of BC, DE, and FG loop residues of the monobody are in cyan, yellow, and green, respectively. The oxygen and nitrogen atoms are shown in red and blue, respectively. The monobody backbone is also shown as a transparent cartoon model. (B) Interactions between the monobody FG loop residues (stick models) and the MBP bottom lobe epitope (shown as surfaces). The surfaces of aromatic residues are shown in yellow. Potential polar interactions for the hydroxyl oxygen atom of the paratope Tyr residues are shown as dashed lines with their distances. The monobody residues are indicated in bold. (C) The interactions in the top lobe epitope. MBP residues are drawn with carbon atoms in gray. The carbon atoms of BC, DE, and FG loop residues of the monobody are in cyan, yellow, and green, respectively. (D) The buried surface areas of the monobody residues. Only those for the binding complex are shown.