Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a "closed" FakB1 state to an "open" state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior. Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a "closed" FakB1 state to an "open" state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior. Lipids are hydrophobic molecules with limited water solubility and must be transferred between membrane organelles or to soluble enzymes by specialized transfer proteins that are able to sequester their apolar cargo and facilitate their transport through the cytosol (1Wong L.H. Copic A. Levine T.P. Advances on the transfer of lipids by lipid transfer proteins.Trends Biochem. Sci. 2017; 42: 516-530Google Scholar, 2Wong L.H. Gatta A.T. Levine T.P. Lipid transfer proteins: The lipid commute via shuttles, bridges and tubes.Nat. Rev. Mol. Cell Biol. 2019; 20: 85-101Google Scholar, 3Hertzel A.V. Bernlohr D.A. The mammalian fatty acid-binding protein multigene family: Molecular and genetic insights into function.Trends Endocrinol. Metab. 2000; 11: 175-180Google Scholar). There are five essential steps common to all lipid transfer processes (Fig. 1A). First, the transfer protein ferries its enclosed cargo to the membrane. Second, it collides with the membrane bilayer surface via an electrostatic attraction or by exploiting a specific membrane ligand such as the phospholipid head group. Third, a conformational change occurs that opens the protein interior, exposes the buried lipid, and allows for its exchange with another lipid in the membrane. A ligand-free binding protein is an obligatory intermediate in the exchange process, and some binding proteins deposit their cargo and disengage from the membrane without loading a new ligand. Fourth, the conformational change reverses and the new protein–lipid complex dissociates from the membrane. Fifth, the complex moves through the cytosol to its destination. There are detailed crystal structures of the lipid transfer proteins in solution that reveal how they sequester lipids from solvent (4Zimmerman A.W. Veerkamp J.H. New insights into the structure and function of fatty acid-binding proteins.Cell. Mol. Life Sci. 2002; 59: 1096-1116Google Scholar, 5Storch J. Corsico B. The emerging functions and mechanisms of mammalian fatty acid-binding proteins.Annu. Rev. Nutr. 2008; 28: 73-95Google Scholar). In contrast, structures of the open exchange state of transfer proteins at the membrane interface remain elusive. The mammalian fatty acid (FA)-binding protein (FABP) family has a common β barrel fold formed by ten antiparallel β-strands that create a large internal cavity to accommodate many different FA structures (3Hertzel A.V. Bernlohr D.A. The mammalian fatty acid-binding protein multigene family: Molecular and genetic insights into function.Trends Endocrinol. Metab. 2000; 11: 175-180Google Scholar, 5Storch J. Corsico B. The emerging functions and mechanisms of mammalian fatty acid-binding proteins.Annu. Rev. Nutr. 2008; 28: 73-95Google Scholar, 6Storch J. Thumser A.E. The fatty acid transport function of fatty acid-binding proteins.Biochim. Biophys. Acta. 2000; 1486: 28-44Google Scholar). The amino termini of FABPs form a helix-loop-helix motif that "caps" the internal cavity of the β barrel. Crystal structures show no obvious opening for an external FA to access the interior pocket, but NMR studies have revealed that the helical cap region is dynamic (5Storch J. Corsico B. The emerging functions and mechanisms of mammalian fatty acid-binding proteins.Annu. Rev. Nutr. 2008; 28: 73-95Google Scholar, 7Herr F.M. Aronson J. Storch J. Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.Biochemistry. 1996; 35: 1296-1303Google Scholar, 8Corsico B. Cistola D.P. Frieden C. Storch J. The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12174-12178Google Scholar). Site-directed mutagenesis studies support a role for the cap helices in membrane association and the subsequent extraction of the FA (6Storch J. Thumser A.E. The fatty acid transport function of fatty acid-binding proteins.Biochim. Biophys. Acta. 2000; 1486: 28-44Google Scholar, 7Herr F.M. Aronson J. Storch J. Role of portal region lysine residues in electrostatic interactions between heart fatty acid binding protein and phospholipid membranes.Biochemistry. 1996; 35: 1296-1303Google Scholar, 8Corsico B. Cistola D.P. Frieden C. Storch J. The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12174-12178Google Scholar, 9Zhang F. Lucke C. Baier L.J. Sacchettini J.C. Hamilton J.A. Solution structure of human intestinal fatty acid binding protein: Implications for ligand entry and exit.J. Biomol. NMR. 1997; 9: 213-228Google Scholar, 10Likic V.A. Prendergast F.G. Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.Protein Sci. 1999; 8: 1649-1657Google Scholar, 11Liou H.L. Storch J. Role of surface lysine residues of adipocyte fatty acid-binding protein in fatty acid transfer to phospholipid vesicles.Biochemistry. 2001; 40: 6475-6485Google Scholar) while molecular dynamics (MD) simulations corroborate the dynamic nature of the two-helix motif in solution (12Bakowies D. van Gunsteren W.F. Simulations of apo and holo-fatty acid binding protein: Structure and dynamics of protein, ligand and internal water.J. Mol. Biol. 2002; 315: 713-736Google Scholar, 13Friedman R. Nachliel E. Gutman M. Fatty acid binding proteins: Same structure but different binding mechanisms? Molecular dynamics simulations of intestinal fatty acid binding protein.Biophys. J. 2006; 90: 1535-1545Google Scholar, 14Tsfadia Y. Friedman R. Kadmon J. Selzer A. Nachliel E. Gutman M. Molecular dynamics simulations of palmitate entry into the hydrophobic pocket of the fatty acid binding protein.FEBS Lett. 2007; 581: 1243-1247Google Scholar, 15Long D. Mu Y. Yang D. Molecular dynamics simulation of ligand dissociation from liver fatty acid binding protein.PLoS One. 2009; 4e6081Google Scholar, 16Li Y. Li X. Dong Z. Exploration of gated ligand binding recognizes an allosteric site for blocking FABP4-protein interaction.Phys. Chem. Chem. Phys. 2015; 17: 32257-32267Google Scholar, 17Matsuoka D. Sugiyama S. Murata M. Matsuoka S. Molecular dynamics simulations of heart-type fatty acid binding protein in apo and holo forms, and hydration structure analyses in the binding cavity.J. Phys. Chem. B. 2015; 119: 114-127Google Scholar, 18Guo Y. Duan M. Yang M. The observation of ligand-binding-relevant open states of fatty acid binding protein by molecular dynamics simulations and a Markov state model.Int. J. Mol. Sci. 2019; 20: 3476Google Scholar). The current model posits that the cap exists in an "open" state when an FABP is bound to the bilayer to allow FA exchange, but the conformation of the bilayer-associated FABP has not been described. The bacterial class of FA transfer proteins is called FakB, and the bound acyl chains are completely enclosed within the protein interior. However, they are structurally distinct from mammalian FABPs and have restricted internal cavities that are tailored to bind only selected FA structures (19Parsons J.B. Broussard T.C. Bose J.L. Rosch J.W. Jackson P. Subramanian C. Rock C.O. Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10532-10537Google Scholar, 20Broussard T.C. Miller D.J. Jackson P. Nourse A. White S.W. Rock C.O. Biochemical roles for conserved residues in the bacterial fatty acid binding protein family.J. Biol. Chem. 2016; 291: 6292-6303Google Scholar, 21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar, 22Gullett J.M. Cuypers M.G. Frank M.W. White S.W. Rock C.O. A fatty acid binding protein of Streptococcus pneumoniae facilitates the acquisition of host polyunsaturated fatty acids.J. Biol. Chem. 2019; 294: 16416-16428Google Scholar). FakB proteins shuttle FA and acyl-PO4 between the membrane and soluble enzymatic partners by undergoing the exchange cycle in Figure 1A (19Parsons J.B. Broussard T.C. Bose J.L. Rosch J.W. Jackson P. Subramanian C. Rock C.O. Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10532-10537Google Scholar, 21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar). Staphylococcus aureus has two FakBs. FakB1 binds saturated FA (19Parsons J.B. Broussard T.C. Bose J.L. Rosch J.W. Jackson P. Subramanian C. Rock C.O. Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10532-10537Google Scholar) and is responsible for the activation of FA arising from phospholipid turnover (21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar, 23Ericson M.E. Subramanian C. Frank M.W. Rock C.O. Role of fatty acid kinase in cellular lipid homeostasis and SaeRS-dependent virulence factor expression in Staphylococcus aureus.mBio. 2017; 8e00988–17Google Scholar). FakB2 selectively binds monounsaturated FA (19Parsons J.B. Broussard T.C. Bose J.L. Rosch J.W. Jackson P. Subramanian C. Rock C.O. Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10532-10537Google Scholar, 20Broussard T.C. Miller D.J. Jackson P. Nourse A. White S.W. Rock C.O. Biochemical roles for conserved residues in the bacterial fatty acid binding protein family.J. Biol. Chem. 2016; 291: 6292-6303Google Scholar) and is involved in the acquisition of host unsaturated FA at infection sites (24Frank M.W. Yao J. Batte J.L. Gullett J.M. Subramanian C. Rosch J.W. Rock C.O. Host fatty acid utilization by Staphylococcus aureus at the infection site.mBio. 2020; 11e00920–20Google Scholar). The FA is integral to the stability of FakB proteins, and mutation of the hydrogen bond network that holds the FA carboxyl in place results in structurally unstable proteins (20Broussard T.C. Miller D.J. Jackson P. Nourse A. White S.W. Rock C.O. Biochemical roles for conserved residues in the bacterial fatty acid binding protein family.J. Biol. Chem. 2016; 291: 6292-6303Google Scholar). Although cellular engagement thermal shift assays indicate that apo-FakB2 exists in cells (21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar), the apo-FakBs are unstable and have not been isolated for biochemical analysis (19Parsons J.B. Broussard T.C. Bose J.L. Rosch J.W. Jackson P. Subramanian C. Rock C.O. Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 10532-10537Google Scholar, 20Broussard T.C. Miller D.J. Jackson P. Nourse A. White S.W. Rock C.O. Biochemical roles for conserved residues in the bacterial fatty acid binding protein family.J. Biol. Chem. 2016; 291: 6292-6303Google Scholar, 21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar, 22Gullett J.M. Cuypers M.G. Frank M.W. White S.W. Rock C.O. A fatty acid binding protein of Streptococcus pneumoniae facilitates the acquisition of host polyunsaturated fatty acids.J. Biol. Chem. 2019; 294: 16416-16428Google Scholar). Like FABPs, the protein conformation that allows the FA to escape into the membrane is unknown. Despite their dissimilar folds, the mammalian and bacterial FA transfer proteins face an identical topological challenge. Namely, a conformational change must occur to create a diffusion channel for the FA to freely exchange between its position in the membrane bilayer and the interior of the transfer protein. Here, we use a combination of X-ray crystallography, NMR spectroscopy, MD simulations, site-directed mutagenesis, and functional biochemical assays to characterize the membrane-bound conformation of FakB. This study focuses on FakB1, an FA transfer protein from S. aureus that binds palmitic acid (16:0) before presenting it to FakA for phosphorylation and exchange with the membrane. NMR dynamics measurements detect a dynamic 23-residue region covering the FakB1 FA binding tunnel. We captured crystal structures of the open FakB1 conformation by introducing point mutations that insert bulky residues into the narrow FA tunnel. The open conformation arises from a conformational change in the dynamic region that rotates helix α8 outward to uncover the FA-binding tunnel and reorganizes the adjacent α8-β9 loop into a new amphipathic helix. MD simulations reveal how the newly formed helix α8′ inserts into the phospholipid bilayer to create a diffusion path for the FA to exit into the membrane. Site-directed mutagenesis coupled with biochemical assays corroborates the roles of key residues in this process. These data provide an understanding of how a conformational change in FakB1 facilitates the exchange of FA between the protein interior and the membrane that provides key insights into how this process occurs in mammalian FA binding proteins. FakB crystal structures show that the FA is completely buried within the protein interior with only the FA carboxylate group exposed for phosphorylation (20Broussard T.C. Miller D.J. Jackson P. Nourse A. White S.W. Rock C.O. Biochemical roles for conserved residues in the bacterial fatty acid binding protein family.J. Biol. Chem. 2016; 291: 6292-6303Google Scholar, 21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar, 22Gullett J.M. Cuypers M.G. Frank M.W. White S.W. Rock C.O. A fatty acid binding protein of Streptococcus pneumoniae facilitates the acquisition of host polyunsaturated fatty acids.J. Biol. Chem. 2019; 294: 16416-16428Google Scholar). The structures also suggest a potential focal point for the conformational exchange that must occur to release the enclosed FA. The bulk of FakB1 forms the acyl chain binding pocket with the region between Asp164 and Lys186 forming a lid over the first eight carbons of the FA chain (Fig. 1B). Residues 175 to 186 are generally poorly resolved in the many deposited FakB structures, and in FakB1, this region has the highest B-factors (21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar) suggesting that these residues are conformationally flexible. This region is connected to the rest of the protein by a hydrophobic interface and terminates at the bound FA with two hydrogen-bonding interactions between the side chains of Arg173 and Ser171 and the backbone carbonyl of Ile94 and the Nδ1 of His270, respectively (Fig. 1B). We used NMR spectroscopy to independently identify the dynamic regions of FakB1 in solution. The 2D [15N,1H] TROSY spectrum of [15N,13C]FakB1 at 293 K showed well-dispersed resonances, and all but four residues were assigned with the aid of spectra collected at 293 K using [2H,15N,13C]FakB1 (Fig. S1). The secondary structure calculated from TALOS analysis of the NMR spectra correlates well with the secondary structural elements observed in the FakB1 crystal structure (Fig. S2A) with the exception of helix α1 and sheet β5 that are not apparent from the TALOS analysis. Also, residues 178 to 181 within the α8-β9 loop are in an α-helical conformation based on the NMR spectra but have a less pronounced helical nature in the crystal structure. Carr–Purcell–Meiboom–Gill relaxation dispersion (CPMG-RD) spectra were acquired using [2H,15N,13C]FakB1, at two different field strengths (81.1 MHz and 60.8 MHz) at 293 K to map the mobility within the FakB1 structure. The 15N relaxation dispersion profiles revealed that FakB1 is stably folded but ten residues adjacent to the bound FA are clearly dynamic (Fig. 1C). Four of these residues are within the Asp164-Lys186 region and include the central Arg173 and Leu165 at the amino-terminus of the region. The datasets were a close fit to a two-state exchange model using the equation of Carver and Richards (25Carver J.P. Richards R.E. General 2-site solution for chemical exchange produced dependence of T2 upon Carr-Purcell Pulse separation.J. Magn. Reson. 1972; 6: 89-105Google Scholar) (Fig. 1D). Data from all ten residues were used to fit a global two-state model with forward (kf) and reverse (kr) exchange rate constants 12.0 ± 1.0 s−1 and 753.0 ± 53.0 s−1, respectively, yielding an exchange rate constant (kex = kf + kr) of 764.2 ± 53.0 s−1. A Gibbs free energy barrier of 2.41 ± 0.07 kcal/mol is calculated from the exchange rates for the FakB1 conformational change. The major (98.5%) and the minor (1.5%) conformations exchange on the millisecond timescale. The rotation of the dynamic Arg173 residue away from the protein is predicted to expose and promote the opening of a portal to the buried FA. We determined the X-ray structure of FakB1(R173A) to determine if this was the case (Fig. 1E), but its overall structure was identical to that of FakB1 (RMSD = 0.210 Å) (Fig. S3A). The hydrophobic interface and the Ser171-His270 interaction are apparently sufficient to maintain the connection between the dynamic region and the body of FakB1 even though the absence of Arg173 does expose the first eight carbons of the FA to solvent as predicted (Fig. 1E). FakB1(R173A) is a few degrees less stable than FakB1 consistent with the loss of the hydrogen bond connection to Ile94 (Fig. S3B), and as expected (20Broussard T.C. Miller D.J. Jackson P. Nourse A. White S.W. Rock C.O. Biochemical roles for conserved residues in the bacterial fatty acid binding protein family.J. Biol. Chem. 2016; 291: 6292-6303Google Scholar), FakB1(R173A) was catalytically inactive. Unlike the mammalian FABPs, FakB1 has a tight binding tunnel that presents an alternative opportunity to induce the conformational change by using site-directed mutagenesis to modulate the optimal packing of the FA into its binding site. We identified two alanine residues (121 and 158) in the tunnel and mutated them to bulkier residues to partially occlude the FA-binding pocket and push the FA out of its pocket to destabilize the fully closed FakB1 conformation (Fig. S3C). The FakB1(A121I) and FakB1(A158L) mutant proteins were fully active in FA kinase assays with the same apparent affinity for FakA (Fig. 1F), and analytical ultracentrifugation verified that both continue to form tight complexes with FakA (Table S1). However, the mutations did alter two key properties of FakB1. First, compared with the wild-type protein, the thermal stabilities of FakB1(A121I) and FakB1(A158L) were reduced by 6 °C and 4 °C, respectively (Fig. 1G), showing that the mutations indeed prevent FakB1 from adopting its most stable conformation. Second, the mutations altered the FA selectivity of FakB1 in a physiological setting. When expressed in Escherichia coli, the total amount of FA incorporated by the FakB1 mutant proteins was the same as the wild-type protein, but FakB1 only supported the incorporation of palmitic acid (16:0), whereas both mutant proteins were less specific and also incorporated oleate (18:1) and linoleate (18:2) (Fig. 1H). These data show that the tunnel mutations alter the stability and selectivity of FakB1, but do not impair the overall function of FakB1 in vivo or in vitro. The crystal structure of FakB1(A121I) was determined at 2.02 Å resolution (Table 1). We observed two molecules (molA and molB) in different conformational states in the asymmetric unit. The prototypical FakB1 closed conformation was exemplified by molB, but in molA, the dynamic region undergoes a significant structural rearrangement (Fig. 2A). Specifically, Arg173 disengages from the FA and Ile94, helix α8 (Leu165 to Ser171) moves away from the protein, and residues 175 to 186 form a new α-helix that rotates away from the protein by ∼180° in concert with the movement of helix α8. Excluding the structural rearrangements observed in FakB1(A121I), the open and closed structures superimpose with an RMSD of 0.356 Å showing that the conformational change is confined to these residues (Fig. 2A). Notably, the conformational change occurs precisely in the region of the protein revealed by NMR spectroscopy to exhibit dynamic properties (Fig. 1C). The surface rendering of FakB1(A121I) shows that the conformational change exposes the ligand-binding cavity and creates a portal to the FA (Fig. 2B).Table 1X-ray crystallography data collection, refinement, and validation statisticsComplexFakB1(A121I)-palmitate (open)FakB1(A158L)-myristate (open)FakB1(R173A)-palmitate (closed)FakB1(A121I, A158L)-Palmitate (open)PDB codes6MH96NM17SCL7SG3Data collection Beamline (APS)SER-CAT 22-IDSER-CAT 22-IDSER-CAT 22-IDSER-CAT 22-ID Temperature (K)100100100100 Space groupP1P1P1P1 Cell dimensionsa, b, c (Å)33.5, 53.9, 86.033.4, 53.5, 86.233.2, 53.9, 84.433.2, 53.6, 85.9α, β, γ (°)103.7, 90.4, 107.376.9, 89.4, 72.3104.3, 90.7, 107.5104.2, 90.5, 107.0 Resolution (Å)83.29–2.0283.74–2.3349.60–1.6049.46–2.35 Rsym or Rmerge0.066 (0.726)aValues in parentheses are for highest-resolution shell.0.086 (0.813)0.056 (0.596)0.069 (0.492) Rpim0.039 (0.438)0.051 (0.475)0.033 (0.351)0.042 (0.298) Unique reflections34,458 (2509)23,124 (2250)63,745 (2951)20,941 (1997) Redundancy3.8 (3.7)3.9 (3.9)3.8 (3.8)3.6 (3.6) CC (1/2)0.997 (0.724)0.998 (0.748)0.998 (0.841)0.997 (0.867) Mean I/σI12.3 (1.9)10.2 (1.9)7.0 (2.0)6.9 (1.9) Completeness (%)94.4 (91.3)98.2 (97.7)89.7 (83.5)92.0 (90.7) Wilson B-factor (Å−2)34.838.024.042.3Model quality Rwork/Rfree20.7/25.823.7/28.816.19/19.3121.17/25.09 No. atomsProtein4539448344264188Ligand/ion36324836Water20713435969 B-factorsProtein41.753.639.958.2Fatty acid36.048.735.161.3Water46.351.448.053.6 R.m.s. deviationsBond lengths (Å)0.0020.0020.0080.003Bond angles (°)0.4490.4771.0220.500 Ramachandran plotFavored (%)97.092.196.897.2Allowed (%)2.76.02.52.0Outliers (%)0.41.90.71.3Clashscore5.77.85.46.7a Values in parentheses are for highest-resolution shell. Open table in a new tab We also determined the 2.33 Å crystal structure of FakB1(A158L) (Table 1), which was structurally identical to FakB1(A121I). The open FakB1(A158L) molA structure superimposes on the FakB1(A121I) molA structure with an RMSD of 0.265 Å (Fig. 2C). In FakB1(A121I), the bulkier side chain displaces the middle of the aliphatic chain of the FA by 0.9 Å and its distal end by ∼1.4 Å that, in turn, shifts the tunnel residues Val162, Leu191, and Leu271 and pushes against the dynamic region. A similar process occurs in FakB1(A158L). The 2.35 Å crystal structure of the FakB1(A121I, A158L) double mutant was also determined (Table 1), and superposition of the FakB1(A121I) and FakB1(A121I, A158L) structures shows that they are basically identical with an RMSD = 0.241 Å (Fig. S3D). Thus, three independent mutants generated the identical conformational change. The FakB1(A121I) is the highest-resolution crystal structure and was used as the representative open conformation for further analysis. The dynamic region between residues 177 and 186 is generally poorly resolved or not present in many FakB structures deposited in the PDB. In our FakB1 structure (21Cuypers M.G. Subramanian C. Gullett J.M. Frank M.W. White S.W. Rock C.O. Acyl chain selectivity and physiological roles of Staphylococcus aureus fatty acid binding proteins.J. Biol. Chem. 2019; 294: 38-49Google Scholar), residues 180 to 183 have the highest B-factors in the crystal structure, but the positions of the residues in the α8-β9 loop can be located in the 2Fo-Fc and SA-OMIT electron density maps (Fig. S4A). In the FakB1(A121I) structure, the α8-β9 loop residues become helix α8′, and the residues within this helix have lower B-factors than in the FakB1 structure (Fig. S4B). This allows the conformation of the new 12-residue helix α8′ to be clearly defined in the 2Fo-Fc and SA-OMIT maps (Fig. S4B). In the closed conformation, Trp180 is tightly packed against the hydrophobic interior of the protein, and Arg173 is packed onto the bound FA (Fig. 2D, left panel). There is also a hydrogen bond network consisting of Thr62, Ser95, Ser171, His270, and the FA carboxylate that appears to balance the negative charge on the FA (Fig. 2D, right panel). In the open conformation, Arg173 becomes disengaged from FakB1 (Fig. 2E, left panel), and the newly formed 12-residue helix that we designate as α8′ has a distinct exposed hydrophobic surface consisting of Ala177, Trp180, Val181, Leu184, and Leu185. We suggest that the low dielectric constant within the FakB1(A121I) crystal lattice coupled with the location of Phe38 and Ile44 on the neighboring molecule that is only available to molA create an apolar environment for the relocation and stabilization of helix α8′ (Fig. S5A). We also note that the disengagement of Arg173 from the FA and the outward movements of α8 and α8′ disrupt the hydrogen bond network surrounding the FA carboxylate by breaking the key Ser171-His270 hydrogen bond and exposing His270 to solvent (Fig. 2E, right panel). FakB1(A121I) was analyzed by NMR to determine how the mutation impacts protein dynamics. The 2D [15N,1H] TROSY spectrum of FakB1(A121I) was similar to that of the wild-type protein (Fig. S5B) with the largest chemical shift differences localized to the area adjacent to the Ala121 mutation (Fig. S5C). CPMG-RD data for [2H,15N,13C]FakB1(A121I) collected at two field strengths at 293 K revealed 12 residues with 15N relaxation dispersion profiles that mapped to the same locations as the ten dynamic residues in FakB1 (Fig. S5D). These residues are more mobile than in FakB1(121I) as exemplified by Arg173 and Leu165 (compare Figs. S5E and 1D). The global exchange rate was kex = 757.3