Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes

Kühlmann, Ulrike C.; Pommer, Ansgar J.; Moore, Geoffrey R.; James, Richard; Kleanthous, Colin

doi:10.1006/jmbi.2000.3945

Cited by 145 publications

(228 citation statements)

References 57 publications

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“…A similar conclusion has been reached by Kleanthous et al (35)(36)(37). Through studies of DNase binding by the immunity proteins they have shown that conserved residue hot spots (from helix III) act as a binding site anchor, whereas the variable residues (from helix II) define specificity.…”

Section: Resultssupporting

confidence: 68%

Protein–protein interactions: Structurally conserved residues distinguish between binding sites and exposed protein surfaces

Elkayam

Wolfson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

558

500

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Polar residue hot spots have been observed at protein-protein binding sites. Here we show that hot spots occur predominantly at the interfaces of macromolecular complexes, distinguishing binding sites from the remainder of the surface. Consequently, hot spots can be used to define binding epitopes. We further show a correspondence between energy hot spots and structurally conserved residues. The number of structurally conserved residues, particularly of high ranking energy hot spots, increases with the binding site contact size. This finding may suggest that effectively dispersing hot spots within a large contact area, rather than compactly clustering them, may be a strategy to sustain essential key interactions while still allowing certain protein flexibility at the interface. Thus, most conserved polar residues at the binding interfaces confer rigidity to minimize the entropic cost on binding, whereas surrounding residues form a flexible cushion. Furthermore, our finding that similar residue hot spots occur across different protein families suggests that affinity and specificity are not necessarily coupled: higher affinity does not directly imply greater specificity. Conservation of Trp on the protein surface indicates a highly likely binding site. To a lesser extent, conservation of Phe and Met also imply a binding site. For all three residues, there is a significant conservation in binding sites, whereas there is no conservation on the exposed surface. A hybrid strategy, mapping sequence alignment onto a single structure illustrates the possibility of binding site identification around these three residues.protein-protein interfaces ͉ hot spots ͉ molecular recognition ͉ binding site prediction ͉ residue conservation R inge (1) has raised the question ''what makes a binding site a binding site?'' Many studies have addressed this intriguing and vastly important problem. Being able to a priori predict binding sites would both limit the conformational search in drug design, facilitate the prediction of protein-protein interactions (2), and may provide leads to binding site design.A number of studies have examined the attributes of proteinbinding sites (3-5). Although binding sites on enzyme surfaces typically consist of a concave cleft shape (6, 7) and similarly small ligand binding sites on receptor surfaces (8), this is not the case for the larger protein-protein complexes (9-12). Enzyme-binding sites were shown to frequently be the largest cavities on the enzyme surface (6, 7). On the other hand, the shape of dimer-binding sites is usually quite flat (9) and practically indistinguishable from other patches on the protein surface. Native binding sites do not yield the largest possible interfaces between two protein molecules. A docking study has shown that nonnative interfaces can be larger, and bury a larger extent of total or nonpolar surface areas (13). A similar observation has been made for the number of salt bridges or hydrogen bonds (13,14). Hence, although interfaces are frequently largely hydrophobic ...

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Section: Resultssupporting

confidence: 68%

Protein–protein interactions: Structurally conserved residues distinguish between binding sites and exposed protein surfaces

Elkayam

Wolfson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

558

500

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“…E9 DNase Y83 is on the edge of the interface, which in the cognate complex hydrogen bonds directly to T27 and S29 in loop I of Im9 (31), clamping the loop to the interface and ensuring optimal alignment of Im helix II and DNase specificity sites (Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

The structural and energetic basis for high selectivity in a high-affinity protein-protein interaction

Meenan

Sharma

Fleishman

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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High-affinity, high-selectivity protein-protein interactions that are critical for cell survival present an evolutionary paradox: How does selectivity evolve when acquired mutations risk a lethal loss of high-affinity binding? A detailed understanding of selectivity in such complexes requires structural information on weak, noncognate complexes which can be difficult to obtain due to their transient and dynamic nature. Using NMR-based docking as a guide, we deployed a disulfide-trapping strategy on a noncognate complex between the colicin E9 endonuclease (E9 DNase) and immunity protein 2 (Im2), which is seven orders of magnitude weaker binding than the cognate femtomolar E9 DNase-Im9 interaction. The 1.77 Å crystal structure of the E9 DNase-Im2 complex reveals an entirely noncovalent interface where the intersubunit disulfide merely supports the crystal lattice. In combination with computational alanine scanning of interfacial residues, the structure reveals that the driving force for binding is so strong that a severely unfavorable specificity contact is tolerated at the interface and as a result the complex becomes weakened through "frustration." As well as rationalizing past mutational and thermodynamic data, comparing our noncognate structure with previous cognate complexes highlights the importance of loop regions in developing selectivity and accentuates the multiple roles of buried water molecules that stabilize, ameliorate, or aggravate interfacial contacts. The study provides direct support for dual-recognition in colicin DNase-Im protein complexes and shows that weakened noncognate complexes are primed for high-affinity binding, which can be achieved by economical mutation of a limited number of residues at the interface.colicins | crystallography | disulfide-trapping | specificity | frustration S pecificity in protein-protein interactions (PPIs) is critical for the organization of macromolecular complexes involved in all aspects of cellular homeostasis and differentiation. Within the vast interaction networks in cells there is significant redundancy, with many proteins acting as "hubs" that recognize multiple binding partners (1), and yet also significant discrimination (2). The factors that tip the balance in favor of specificity or promiscuity in PPIs while not clear are coming under increasing scrutiny (3). Understanding this balance is critical both from fundamental and applied perspectives. Protein therapeutics are finding increasing use in medicine but off-target effects can have disastrous consequences (4). High-affinity, high-selectivity binding is therefore an essential goal in such engineered platforms. Advances in defining the molecular and thermodynamic basis for binding affinity have been made in a multitude of natural and engineered/designed PPIs (5-7), but our molecular knowledge of specificity remains rudimentary. In large part this is due to the lack of highresolution structural information on weak and transient proteinprotein complexes that are evolutionarily related to a cognate hig...

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“…The toxic 15-kDa C-terminal DNase domain of E9 forms a tight hetero-dimeric complex with its cognate 9.5 kDa immunity protein Im9 with a dissociation constant of ϳ10 Ϫ16 M [15,18]. The molecular determinants of this strong interaction have been studied in-depth by X-ray crystallography and comparative alanine scanning [19]. The DNase domain can be isolated as an active enzyme [20,21] and its structure has been analyzed by X-ray crystallography in presence and absence of its cognate immunity protein Im9 [19].…”

mentioning

confidence: 99%

“…The molecular determinants of this strong interaction have been studied in-depth by X-ray crystallography and comparative alanine scanning [19]. The DNase domain can be isolated as an active enzyme [20,21] and its structure has been analyzed by X-ray crystallography in presence and absence of its cognate immunity protein Im9 [19]. The solution phase structure of free Im9, and bound to E9, has also been studied by NMR [22].…”

mentioning

confidence: 99%

Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

Scholten

Visser

Heuvel

et al. 2006

J. Am. Soc. Mass Spectrom.

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To understand how proteins perform their function, knowledge about their structure and dynamics is essential. Here we use a combination of an efficient chemical lysine acetylation reaction and nanoLC-MALDI tandem mass spectrometry to probe the accessibility of every lysine residue in a protein complex. To demonstrate the applicability of this approach, we studied the interaction between the DNase domain of Colicin E9 (E9) and its immunity protein Im9. Free E9 and E9 in complex with Im9 were rapidly acetylated, followed by proteolytic digestion and analysis by LC-MALDI-TOF/TOF MS/MS. Acetylated peptides could be filtered out of the complex peptide mixtures using selective ion chromatograms of the specific immonium marker ions. Additionally, isobaric acetylated peptides, acetylated at different sites, could be separated by their LC retention times. The combination of LC and MALDI-TOF/TOF MS/MS provided information about the amount of acetylation on each individual lysine even for peptides containing several lysine residues. In general, our data agree well with those derived from the crystal structure of E9 and the E9:Im9 complex. Interestingly, next to in the binding interface expected lysines, K89 and K97, two from the crystal structure data unexpected lysines, K81 and K76, were observed to become less exposed upon Im9 binding. Moreover, K55 and K63, positioned in the predicted DNA binding region, were also found to be less accessible upon Im9 binding. These findings may illustrate some of the described differences in the solution-phase structure of the E9:Im9 complex compared with the crystal structure. (J Am Soc Mass Spectrom 2006, 17, 983-994)

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Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes

Cited by 145 publications

References 57 publications

Protein–protein interactions: Structurally conserved residues distinguish between binding sites and exposed protein surfaces

Protein–protein interactions: Structurally conserved residues distinguish between binding sites and exposed protein surfaces

The structural and energetic basis for high selectivity in a high-affinity protein-protein interaction

Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

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