On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

Abstract

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.

Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein, HisJ

“…Additionally, our ITC data confirm the ligand binding abilities towards all four ligands reported earlier . Moreover, we also show that HisJ interacts selectively with l ‐His over d ‐histidine and is capable of binding to some biologically relevant l ‐His analogs, such as 1‐methyl‐ l ‐His and 3‐methyl‐ l ‐His.…”

“…In our 1 H,N HSQC NMR titration experiments with the HisJ protein we observe that all the resonances are affected in a similar manner by additions of L‐His, L‐Arg, L‐Lys, and L‐Orn. Interestingly, resonances arising from multiple residues from different parts of the protein are affected upon ligand addition, suggesting an overall conformational change for the protein rather than a highly localized binding event.…”

“… 1 H,N (TROSY) HSQC overlay spectra of D,N HisJ in the presence of an excess of 16 times l ‐His (black), l ‐Arg (red), l ‐Lys (green), and l ‐Orn (blue).…”

“…Subsequently, it binds with HisJ with very high affinity (nM K d ) and translocation of the L‐histidine into the cytosol takes place using the inner membrane ATPase‐permease complex HisQMP 2 . The Lysine, Arginine, Ornithine Binding protein (LAOBP) is a closely related PBP which shares very high sequence identity (70%) with HisJ and it binds L‐lysine, L‐arginine and L‐ornithine in the periplasm with similar high affinity . Interestingly, LAOBP also uses the same inner membrane ATPase‐permease complex as HisJ to transport its ligands into the cytosol .…”

“…Interestingly, LAOBP also uses the same inner membrane ATPase‐permease complex as HisJ to transport its ligands into the cytosol . Previous work has shown that L‐His binds with high affinity (nm) with HisJ and that L‐Lys, L‐Arg and L‐Orn have a high affinity for LAOBP . Additionally, it has also been demonstrated that these two proteins can bind with each other's preferred ligands with μM affinities .…”