NMR Structure and Dynamics of the Engineered Fluorescein-Binding Lipocalin FluA Reveal Rigidification of β-Barrel and Variable Loops upon Enthalpy-Driven Ligand Binding

Abstract

The NMR structure of the 21 kDa lipocalin FluA, which was previously obtained by combinatorial design, elucidates a reshaped binding site specific for the dye fluorescein resulting from 21 side chain replacements with respect to the parental lipocalin, the naturally occurring bilin-binding protein (BBP). As expected, FluA exhibits the lipocalin fold of BBP, comprising eight antiparallel β-strands forming a β-barrel with an α-helix attached to its side. Comparison of the NMR structure of the free FluA with the X-ray structures of BBP•biliverdin IX γ and FluA•fluorescein complexes revealed significant conformational changes in the binding pocket, which is formed by four loops at the open end of the β-barrel as well as adjoining β-strand segments. An 'induced fit' became apparent for the side-chain conformations of Arg 88 and Phe 99, which contact the bound fluorescein in the complex and undergo concerted rearrangement upon ligand binding. Moreover, slower internal motional modes of the polypeptide backbone were identified by measuring transverse 15 N backbone spin relaxation times in the rotating frame for the free FluA and also the FluA•fluorescein complex. A reduction of such motions was detected upon complex formation, indicating rigidification of the protein structure and loss of conformational entropy. This hypothesis was confirmed by isothermal titration calorimetry, showing that ligand binding is enthalpy driven, thus overcompensating negative entropy associated with both ligand binding per se and rigidification of the protein. Our investigation of the solution structure and dynamics as well as thermodynamics of lipocalin-ligand interaction does not only provide insight into the general mechanism of small molecule accommodation in the deep and narrow cavity of this abundant class of proteins but will also support the future design of corresponding binding proteins with novel specificities, so-called "anticalins". NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript sequence homology is low (usually only 10-20%), lipocalins share a common fold comprising an eight-stranded antiparallel β-barrel with an attached α-helix (Figure 1). On one end, the β-barrel is "closed" by short loops and densely packed side chains that form the hydrophobic core. On the other end, the β-barrel is usually "open" to the solvent: there, four loops connecting neighboring β-strands form the entrance to the ligand-binding site. Whereas the β-barrel structure is strictly conserved among lipocalins, the loops around the ligand pocket are highly variable regarding length, amino acid sequence and backbone conformation(2). This finding provides a rationale for the diversity of binding specificities observed for this protein family in nature. In this respect, lipocalins may be compared with immunoglobulins, which carry a set of six structurally hypervariable loops on top of a rigid β-sheet sandwich, thus giving rise to a vast repertoire of antigen specificities(3).Based on this notion, the lipocalin scaffold w...

The structural basis for high affinity binding of α1-acid glycoprotein to the potent antitumor compound UCN-01

“…Addition of a 2-fold excess of UCN-01 to AGP2-FL, however, yielded a much improved 1 H-15 N TROSY-HSQC spectrum, with a higher proportion of the expected number of resonances and more uniform peak homogeneity. AGP2-FL was therefore most likely correctly folded and UCN-01 was able to bind and stabilise the structure as has been observed in other lipocalins (27). The NMR spectrum, however, remained of less than satisfactory quality (Supporting Information Figure 1).…”

Interaction Studies of the Human and Arabidopsis thaliana Med25-ACID Proteins with the Herpes Simplex Virus VP16- and Plant-Specific Dreb2a Transcription Factors

“…All these attributes determine the energetics of the protein-interfaces and the free energy contributions have to be kept in balance for stable complex formation. This process involves a balance between entropy and enthalpy changes where the unfavorable changes might be compensated in the regions that are not part of the binding site [64] – [67] , which might explain the differences in binding energetics between hMed25-ACID/Dreb2a and aMed25-ACID/Dreb2a. Therefore, even though the plant-specific transcription factor Dreb2a is able to bind to the hMed25-ACID, it might still give rise to a different and specific functional fold upon complex-formation with the aMed25-ACID which might be required for proper functionality during activation of transcription in A.thaliana .…”

“…The regions in VP16-TAD which have propensity to form α-helices upon interaction with its target are indicated (435–450 and 465–485) [37] . The black box indicates the region of the nine-amino-acid sequence that has previously been reported as important for interaction of VP16-TAD with several co- factors [41] , [66] and which also has been identified in a range of transcription factors such as VP16, p53, HSF1, NF-kB and NFAT1 [30] , [42] , [67] . The Dreb2a sequence has six identical amino acids that align to that region in VP16-TAD (DFDLDMLGD).…”

Survey of the year 2009: applications of isothermal titration calorimetry

“…In 2009 we identified more than 400 articles after searching Web of Science, PubMed, SciDir and OVID databases using ‘isothermal AND titration AND calorimetry’ or ITC or ‘Isothermal Titration Calorimetry’ search terms. These have been classified into the following categories: Pre‐2009 references cited in the text and review articles 1–43 Protein‐protein and protein‐peptide interactions 44–124 Protein/peptide‐small molecule interactions 125–218 Protein/peptide‐metal ion interactions 219–253 Protein/peptide‐nucleic acid interactions 254–273 Protein/peptide‐lipid interactions 274–292 Protein/peptide‐polysaccharide interactions 293–316 Nucleic acid‐small molecule interactions 317–348 Small molecule interactions …”