The effect of charge reversal mutations in the α-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs,lysophospholipids and bile acids

Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of -helix II. The -helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on -helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the -helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group.     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins

Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family.     

Two fatty acid‐binding proteins expressed in the intestine interact differently with endocannabinoids

Two different members of the fatty acid‐binding protein (FABP) family are found in enterocyte cells of the gastrointestinal system, namely liver‐type and intestinal fatty acid‐binding proteins (LFABP and IFABP, also called FABP1 and FABP2, respectively). Striking phenotypic differences have been observed in knockout mice for either protein, for example, high fat‐fed IFABP‐null mice remained lean, whereas LFABP‐null mice were obese, correlating with differences in food intake. This finding prompted us to investigate the role each protein plays in directing the specificity of binding to ligands involved in appetite regulation, such as fatty acid ethanolamides and related endocannabinoids. We determined the binding affinities for nine structurally related ligands using a fluorescence competition assay, revealing tighter binding to IFABP than LFABP for all ligands tested. We found that the head group of the ligand had more impact on binding affinity than the alkyl chain, with the strongest binding observed for the carboxyl group, followed by the amide, and then the glycerol ester. These trends were confirmed using two‐dimensional ¹H–¹⁵N nuclear magnetic resonance (NMR) to monitor chemical shift perturbation of the protein backbone resonances upon titration with ligand. Interestingly, the NMR data revealed that different residues of IFABP were involved in the coordination of endocannabinoids than those implicated for fatty acids, whereas the same residues of LFABP were involved for both classes of ligand. In addition, we identified residues that are uniquely affected by binding of all types of ligand to IFABP, suggesting a rationale for its tighter binding affinity compared with LFABP.     

Natural ligand binding and transfer from liver fatty acid binding protein (LFABP) to membranes

Liver fatty acid-binding protein (LFABP) is distinctive among fatty acid-binding proteins because it binds more than one molecule of long-chain fatty acid and a variety of diverse ligands. Also, the transfer of fluorescent fatty acid analogues to model membranes under physiological ionic strength follows a different mechanism compared to most of the members of this family of intracellular lipid binding proteins. Tryptophan insertion mutants sensitive to ligand binding have allowed us to directly measure the binding affinity, ligand partitioning and transfer to model membranes of natural ligands. Binding of fatty acids shows a cooperative mechanism, while acyl-CoAs binding presents a hyperbolic behavior. Saturated fatty acids seem to have a stronger partition to protein vs. membranes, compared to unsaturated fatty acids. Natural ligand transfer rates are more than 200-fold higher compared to fluorescently-labeled analogues. Interestingly, oleoyl-CoA presents a markedly different transfer behavior compared to the rest of the ligands tested, probably indicating the possibility of specific targeting of ligands to different metabolic fates.     

Testing of the portal hypothesis: analysis of a V32G, F57G, K58G mutant of the fatty acid binding protein of the murine adipocyte

The portal region of fatty acid binding proteins is hypothesized to function as a dynamic aperture, controlling accessibility of external ligands to the internal fatty acid binding cavity. To test this hypothesis, a triple mutant of the murine FABP4 has been developed (V32G, F57G, K58G, referred to as the portal mutant) that is predicted to constitutively enlarge the opening due to a reduction in the molecular dimensions of the side chains of key portal amino acids. The portal mutant was purified from expressing Escherichia coli, its stability was evaluated, and the thermodynamics and kinetics of ligand binding were compared to that of wild-type protein. Introduction of the three amino acid substitutions caused no significant change in the stability of the protein with a free energy of unfolding of 13.7 kJ/mol as compared to 14.0 kJ/mol for the wild-type protein. The portal mutant exhibited a modest decrease (4-fold) in ligand binding affinity using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) as a surrogate ligand. 1,8-ANS displacement assays revealed that the binding affinity for oleate increased from a K0.5 of 196 +/- 15 nM for the wild-type protein to 165 +/- 8 for the portal mutant, while that for arachidonate decreased from the wild type of 186 +/- 11 nM to 418 +/- 26 nM for the portal mutant. To evaluate cavity accessibility, rate of 1,8-ANS binding was assessed between the portal and wild-type protein. Using equimolar amounts of ligand and protein at 4 degrees, 1,8-ANS bound within the cavity to 95% saturation (t0.95) in 750 ms, while the mutant protein was fully modified in less than 1.4 ms. To independently evaluate cavity accessibility, modification of the sole protein cysteine residue, C117 residing within the cavity near C2-C4 of the bound ligand, was monitored using 5,5'-dithio-bis(2-nitrobenzoic acid) modification. The half time for modification (t0.5) for the wild-type protein was approximately 20 s, while that for V32G F57G K58G occurred in less than a second. As such, enlargement of the portal region of FABP4 markedly increased the accessibility of ligands to the cavity while having only modest effects on ligand affinity. Taken together, these data provide support for the portal region hypothesis and suggest dynamic fluctuations in this region regulate cavity access, but not ligand affinity or selectivity.     

Solution-state molecular structure of apo and oleate-liganded liver fatty acid-binding protein

Rat liver fatty acid-binding protein (LFABP) is distinctive among intracellular lipid-binding proteins (iLBPs): more than one molecule of long-chain fatty acid and a variety of diverse ligands can be bound within its large cavity, and in vitro lipid transfer to model membranes follows a mechanism that is diffusion-controlled rather than mediated by protein-membrane collisions. Because the apoprotein has proven resistant to crystallization, nuclear magnetic resonance spectroscopy offers a unique route to functionally informative comparisons of molecular structure and dynamics for LFABP in free (apo) and liganded (holo) forms. We report herein the solution-state structures determined for apo-LFABP at pH 6.0 and for holoprotein liganded to two oleates at pH 7.0, as well as the structure of the complex including locations of the ligands. 1H, 13C, and 15N resonance assignments revealed very similar types and locations of secondary structural elements for apo- and holo-LFABP as judged from chemical shift indices. The solution-state tertiary structures of the proteins were derived with the CNS/ARIA computational protocol, using distance and angular restraints based on 1H-1H nuclear Overhauser effects (NOEs), hydrogen-bonding networks, 3J(HNHA) coupling constants, intermolecular NOEs, and residual dipolar (NH) couplings. The holo-LFABP solution-state conformation is in substantial agreement with a previously reported X-ray structure [Thompson, J., Winter, N., Terwey, D., Bratt, J., and Banaszak, L. (1997) The crystal structure of the liver fatty acid-binding protein. A complex with two bound oleates, J. Biol. Chem. 272, 7140-7150], including the typical beta-barrel capped by a helix-turn-helix portal. In the solution state, the internally bound oleate has the expected U-shaped conformation and is tethered electrostatically, but the extended portal ligand can adopt a range of conformations based on the computationally refined structures, in contrast to the single conformation observed in the crystal structure. The apo-LFABP also has a well-defined beta-barrel structural motif typical of other members of the iLBP protein family, but the portal region that is thought to facilitate ligand entry and exit exhibits conformational variability and an unusual "open cap" orientation with respect to the barrel. These structural results allow us to propose a model in which ligand binding to LFABP occurs through conformational fluctuations that adjust the helix-turn-helix motif to open or close the top of the beta-barrel, and solvent accessibility to the protein cavity favors diffusion-controlled ligand transport.     

Titration and exchange studies of liver fatty acid-binding protein with 13C-labeled long-chain fatty acids

Uniformly (13)C-labeled long-chain fatty acids were used to probe ligand binding to rat liver fatty acid-binding protein (LFABP), an atypical member of the fatty acid-binding protein (FABP) family that binds more than one molecule of long-chain fatty acid, accommodates a variety of diverse ligands, and exhibits diffusion-mediated lipid transport to membranes. Two sets of (1)H-(13)C resonances were found in a titration series of NMR spectra for oleate-LFABP complexes, indicating that two molecules of the fatty acid are situated in the protein cavity. However, no distinct resonances were observed for the excess fatty acid in solution, suggesting that at least one ligand undergoes rapid exchange with oleate in the bulk solution. An exchange rate of 54 +/- 6 s(-1) between the two sets of resonances was measured directly using (13)C z,z-exchange spectroscopy. In light of these NMR measurements, possible molecular mechanisms for the ligand-exchange process are evaluated and implications for the anomalous fatty acid transport mechanism of LFABP are discussed.     

Structural properties of the adipocyte lipid binding protein

The adipocyte lipid binding protein, ALBP (also adipocyte fatty acid binding protein, A-FABP, 422 protein, aP2, and p15 protein), is one of the most studied of the intracellular lipid binding protein family. Here we sequentially compare the different sources of ALBP and describe the idea that one-third of the amino acid side chains near the N-terminal end appear to play a major role in conformational dynamics and in ligand transfer. Crystallographic data for mouse ALBP are summarized and the ligand binding cavity analyzed in terms of the overall surface and conformational dynamics. The region of the proposed ligand portal is described. Amino acid side chains critical to cavity formation and fatty acid interactions are analyzed by comparing known crystal structures containing a series of different hydrophobic ligands. Finally, we address ALBP ligand binding affinity and thermodynamic studies.     

Molecular simulations of β‐lactoglobulin complexed with fatty acids reveal the structural basis of ligand affinity to internal and possible external binding sites

The interaction of saturated fatty acids of different length (C8:0 to C18:0) with β‐lactoglobulin (βLG) was investigated by molecular dynamics simulation and docking approaches. The results show that the presence of such ligands in the hydrophobic central cavity of βLG, known as the protein calyx, determines an enhancement of atomic fluctuations compared with the unliganded form, especially for loops at the entrance of the binding site. Concerted motions are evidenced for protein regions that could favor the binding of ligands. The mechanism of anchoring of fatty acids of different length is similar for the carboxylate head‐group, through electrostatic interactions with the side chains of Lys60/Lys69. The key protein residues to secure the hydrocarbon chain are Phe105/Met107, which adapt their conformation upon ligand binding. In particular, Phe105 provides an additional hydrophobic clamp only for the tail of the two fatty acids with the longest chains, palmitic, and stearic acid, which are known to bind βLG with a high affinity. The search of additional external binding sites for fatty acids, distinct from the calyx, was also carried out for palmitic acid. Two external sites with a lower affinity were identified as secondary sites, one consisting in a hydrophobic cavity allowing two distinct binding modes for the fatty acid, and the other corresponding to a surface crevice close to the protein α‐helix. The overall results provide a comprehensive picture of the dynamical behavior of βLG in complex with fatty acids, and elucidate the structural basis of the binding of these physiological ligands. Proteins 2014; 82:2609–2619. © 2014 Wiley Periodicals, Inc.     

Liver Fatty Acid-binding Protein Binds Monoacylglycerol in Vitro and in Mouse Liver Cytosol

Liver fatty acid-binding protein (LFABP; FABP1) is expressed both in liver and intestinal mucosa. Mice null for LFABP were recently shown to have altered metabolism of not only fatty acids but also monoacylglycerol, the two major products of dietary triacylglycerol hydrolysis (Lagakos, W. S., Gajda, A. M., Agellon, L., Binas, B., Choi, V., Mandap, B., Russnak, T., Zhou, Y. X., and Storch, J. (2011) Am. J. Physiol. Gastrointest. Liver Physiol. 300, G803–G814). Nevertheless, the binding and transport of monoacylglycerol (MG) by LFABP are uncertain, with conflicting reports in the literature as to whether this single chain amphiphile is in fact bound by LFABP. In the present studies, gel filtration chromatography of liver cytosol from LFABP^−/− mice shows the absence of the low molecular weight peak of radiolabeled monoolein present in the fractions that contain LFABP in cytosol from wild type mice, indicating that LFABP binds sn-2 MG in vivo. Furthermore, solution-state NMR spectroscopy demonstrates two molecules of sn-2 monoolein bound in the LFABP binding pocket in positions similar to those found for oleate binding. Equilibrium binding affinities are ∼2-fold lower for MG compared with fatty acid. Finally, kinetic studies examining the transfer of a fluorescent MG analog show that the rate of transfer of MG is 7-fold faster from LFABP to phospholipid membranes than from membranes to membranes and occurs by an aqueous diffusion mechanism. These results provide strong support for monoacylglycerol as a physiological ligand for LFABP and further suggest that LFABP functions in the efficient intracellular transport of MG.     

Transporter-to-trap conversion: a disulfide bond formation in cellular retinoic acid binding protein I mutant triggered by retinoic acid binding irreversibly locks the ligand inside the protein

Transport proteins must bind their ligands reversibly to enable release at the point of delivery, while irreversible binding is usually associated with the extreme cases of ligand sequestration. Protein conformational dynamics is an important modulator of binding kinetics, as increased flexibility in the regions adjacent to the binding site may facilitate both association and dissociation processes. Ligand entry to, and exit from, the internal binding site of the cellular retinoic acid binding protein I (CRABP I) occurs via a flexible portal region, which functions as a dynamic aperture. We designed and expressed a CRABP I mutant (A35C/T57C), in which a small-scale conformational switch caused by the ligand binding event triggers formation of a disulfide bond in the portal region, thereby arresting structural fluctuations and effectively locking the ligand inside the binding cavity. At the same time, no formation of the disulfide bond is observed in the apo form of the mutant, and most characteristics of the mutant, including protein stability, are very similar to those of the wild-type protein in the absence of retinoic acid. The mutation does not alter the kinetics of retinoic acid binding to the protein, although the disulfide formation makes the binding effectively irreversible, as suggested by the absence of retinoic acid transfer from the holo form of the mutant to lipid vesicles in the absence of a reducing agent. Taken together, these data suggest that the disulfide bond formation in the portal region arrests large-scale structural fluctuations, which are required for retinoic acid release from the protein. The unique properties of the CRABP I mutant described in this work can be used to inspire and guide a design of nanodevices for multiple tasks ranging from sequestering small-molecule toxins in both tissue and circulation to nutrient deprivation of pathogens.     

Effects of ligand binding on dynamics of fatty acid binding protein and interactions with membranes

Intracellular transport of fatty acids involves binding of ligands to their carrier fatty acid binding proteins (FABPs) and interactions of ligand-free and -bound FABPs with membranes. Previous studies focused on ligand-free FABPs. Here, our amide hydrogen exchange data showed that oleic acid binding to human intestinal FABP (hIFABP) stabilizes the protein, most likely through enhancing the hydrogen-bonding network, and induces rearrangement of sidechains even far away from the ligand binding site. Using NMR relaxation techniques, we found that the ligand binding affects not only conformational exchanges between major and minor states but also the affinity of hIFABP to nanodiscs. Analyses of the relaxation and amide exchange data suggested that two minor native-like states existing in both ligand-free and -bound hIFABPs originate from global “breathing” motions, while one minor native-like state comes from local motions. The amide hydrogen exchange data also indicated that helix αII undergoes local unfolding through which ligands can exit from the binding cavity.     

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.     

Model of β-Sheet of Muscle Fatty Acid Binding Protein of Locusta migratoria Displays Characteristic Topology

The β-sheet of muscle fatty acid binding protein of Locusta migratoria (Lm-FABP) was modeled by employing 2-D NMR data and the Rigid Body Assembly method. The model shows the β-sheet to comprise ten β-strands arranged anti-parallel to each other. There is a β-bulge between Ser 13 and Gln 14 which is a difference from the published structure of β-sheet of bovine heart Fatty Acid Binding Protein. Also, a hydrophobic patch consisting of Ile 45, Phe 51, Phe 64 and Phe 66 is present on the surface which is characteristic of most Fatty Acid Binding Proteins. A “gap” is present between βD and βE that provides evidence for the presence of a portal or opening between the polypeptide chains which allows ligand fatty acids to enter the protein cavity and bind to the protein.     

Liver fatty acid binding protein: species variation and the accommodation of different ligands

The crystal structure of rat liver fatty acid binding protein (LFABP) and an alignment of amino acid sequences of all known species have been used to demonstrate two groups or sub-classes. Based on estimates at neutral pH and the electrostatic field calculated using the crystal coordinates, some evidence of changes that occur in going from holo- to apo-forms has been obtained. LFABP belongs to a large family frequently referred to as the intracellular lipid binding proteins or iLBPs. LFABP, unlike other family members, has two fatty acid binding sites. The two cavity sites have been reviewed and arguments for interactions between the sites are presented. Based on the crystal structure of rat LFABP, differences between the A and B groups have been postulated. Last of all, hypothetical models have been built of complexes of LFABP and heme, and LFABP and oleoyl CoA. In both cases, the stoichiometry is one to one and the models show why this is likely.     

A Thermoacidophile-Specific Protein Family,DUF3211, Functions as a Fatty Acid Carrier with Novel Binding Mode

STK_08120 is a member of the thermoacidophile-specific DUF3211 protein family from Sulfolobus tokodaii strain 7. Its molecular function remains obscure, and sequence similarities for obtaining functional remarks are not available. In this study, the crystal structure of STK_08120 was determined at 1.79-Å resolution to predict its probable function using structure similarity searches. The structure adopts an α/β structure of a helix-grip fold, which is found in the START domain proteins with cavities for hydrophobic substrates or ligands. The detailed structural features implied that fatty acids are the primary ligand candidates for STK_08120, and binding assays revealed that the protein bound long-chain saturated fatty acids (>C₁₄) and their trans-unsaturated types with an affinity equal to that for major fatty acid binding proteins in mammals and plants. Moreover, the structure of an STK_08120-myristic acid complex revealed a unique binding mode among fatty acid binding proteins. These results suggest that the thermoacidophile-specific protein family DUF3211 functions as a fatty acid carrier with a novel binding mode.     

The integrity of the alpha-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes

Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes.     

De novo protein fold families expand the designable ligand binding site space

A major challenge in designing proteins de novo to bind user-defined ligands with high affinity is finding backbones structures into which a new binding site geometry can be engineered with high precision. Recent advances in methods to generate protein fold families de novo have expanded the space of accessible protein structures, but it is not clear to what extend de novo proteins with diverse geometries also expand the space of designable ligand binding functions. We constructed a library of 25,806 high-quality ligand binding sites and developed a fast protocol to place (“match”) these binding sites into both naturally occurring and de novo protein families with two fold topologies: Rossman and NTF2. Each matching step involves engineering new binding site residues into each protein “scaffold”, which is distinct from the problem of comparing already existing binding pockets. 5,896 and 7,475 binding sites could be matched to the Rossmann and NTF2 fold families, respectively. De novo designed Rossman and NTF2 protein families can support 1,791 and 678 binding sites that cannot be matched to naturally existing structures with the same topologies, respectively. While the number of protein residues in ligand binding sites is the major determinant of matching success, ligand size and primary sequence separation of binding site residues also play important roles. The number of matched binding sites are power law functions of the number of members in a fold family. Our results suggest that de novo sampling of geometric variations on diverse fold topologies can significantly expand the space of designable ligand binding sites for a wealth of possible new protein functions.