The role of temperature in the binding of the disordered epitope region of human thrombopoietin to antibody: A molecular dynamics simulations study.

The N-terminal domain (163 residues) of Human thrombopoietin (hTPO) is highly conserved and responsible for the receptor-binding. The crystal structure of free hTPO is not yet available, but the crystal structure of its receptor-binding domain (hTPO163) is available in complex with the TN1-Fab antibody. According to a thermodynamic study of hTPO163 binding to TN1-Fab Ab, the ΔH value for binding becomes more negative with an increase in temperature from 283 K to 303 K. The objective of our study is to understand how the free hTPO163 behaves dynamically and to study the effect of temperature on the association of hTPO163 to TN1-Fab antibody through molecular dynamics simulations. We studied the Ag-Ab interactions at two different temperatures 298 K and 303 K. The discontinuous epitope region (residues 98-115) of free hTPO163 displays a conformational switch and it gets stabilized upon binding to the Ab at 303 K. Based on our results, it may be surmised that the epitope region 98-115 is behaving like a disordered epitope. The disordered epitopes are known to be more efficient in binding with the antibody. We also find that, there is an increase in number of hydrogen-bonding interactions and hydrophobic contacts with an increase in the temperature from 298 K to 303 K. Thus, this observation explains a possible reason behind the more negative value of ΔH at the higher temperature 303 K as compared to 298 K.

Inhibition of the activity of HIV-1 protease through antibody binding and mutations probed by molecular dynamics simulations.

HIV-1 protease is an essential enzyme in the life cycle of the HIV-1 virus. The conformational dynamics of the flap region of the protease is critical for the ligand binding mechanism, as well as for the catalytic activity. The monoclonal antibody F11.2.32 raised against HIV-1 protease inhibits its activity on binding. We have studied the conformational dynamics of protease in its free, inhibitor ritonavir and antibody bound forms using molecular dynamics simulations. We find that upon Ab binding to the epitope region (residues 36-46) of protease, the overall flexibility of the protease is decreased including the flap region and the active site, which is similar to the decrease in flexibility observed by inhibitor binding to the protease. This suggests an allosteric mechanism to inhibit protease activity. Further, the protease mutants G40E and G40R are known to have decreased activity and were also subjected to MD simulations. We find that the loss of flexibility in the mutants is similar to that observed in the protease bound to the Ab/inhibitor. These insights highlight the role played by dynamics in the function of the protease and how control of flexibility through Ab binding and site specific mutations can inhibit protease activity.

Conformational dynamics of Ribonuclease Sa and its S48P mutant: Implications for the stability of the mutant protein.

The optimization of ß-turns has been used as a strategy to increase protein thermal stability. One example is the S48P mutation in Ribonuclease Sa, introduced to optimize a ß-turn, which increases the stability of the protein as determined experimentally. Here, we have studied 48SYGY51 ß-turn and its S48P mutant from RNase Sa, as a peptide and as part of the protein, using molecular dynamics simulations. The turn propensity of the region 48SYGY51 shows an increase in both the peptide and protein models on S48P mutation. The mutant protein shows an overall decrease in conformational dynamics and a decrease in conformational heterogeneity as compared to the wildtype protein. A comparatively restricted sampling of the φ-ψ region of GLN47, a pre PRO48 residue, in the mutant protein and some local changes in hydrogen bonding patterns involving residues 20-24 might be contributing to the mutant protein stability. In addition, some long-range hydrogen bonding interactions involving the 60s loop and the salt-bridge interaction involving ASP17-ARG63 could also be contributing to the increase in rigidity and stability of the mutant protein.

Conformational and dynamical basis for cross-reactivity observed between anti HIV-1 protease antibody with protease and an epitope peptide from it.

F11.2.32 is a monoclonal antibody raised against HIV-1 protease and it inhibits protease activity. While the structure of the epitope peptide in complex with the antibody is known, how protease interacts with the antibody is not known. In this study, we model the conformational features of the free and bound epitope peptide and protease-antibody interactions. We find through our simulations, that the free epitope peptide P36-46 samples conformations akin to the bound conformation of the peptide in complex with the Ab, with a ß-turn conformation sampled by the 38LPGR41 sequence highlighting the role of inherent conformational preferences of the peptide. Further, to determine the interactions present between the protease and antibody, we docked the protease in its conformation observed in the crystal structure, onto the antibody and simulated the dynamics of the complex in explicit water. We have identified the key residues involved in hydrogen-bond interactions and salt-bridges in Ag-Ab complex and examined the role of CDR flexibility in binding different conformations of the same epitope sequence in peptide and protein antigens. Thus, our results provide the basis for understanding the cross-reactivity observed between the antibody with protease and the epitope peptide from it.

Mechanism of Initiation, Association, and Formation of Amyloid Fibrils Modeled with the N-Terminal Peptide Fragment, IKYLEFIS, of Myoglobin G-Helix.

Some peptides and proteins undergo self-aggregation under certain conditions, leading to amyloid fibrils formation, which is related to many disease conditions. It is important to understand such amyloid fibrils formation to provide mechanistic detail that governs the process. A predominantly α-helical myoglobin has been reported recently to readily form amyloid fibrils at a higher temperature, similar to its G-helix segment. Here, we have investigated the mechanism of amyloid fibrils formation by performing multiple long molecular dynamics simulations (27 µs) on the N-terminal segment of the G-helix of myoglobin. These simulations resulted in the formation of a single-layered tetrameric ß-sheet with mixed parallel and antiparallel ß-strands and this is the most common event irrespective of many different starting structures. Formation of the single-layered tetrameric ß-sheet takes place following three distinctive pathways. The process of fibril initiation is dependent on temperature. Further, this study provides mechanistic insights into the formation of multilayered fibrilar structure, which could be applicable to a wider variety of peptides or proteins to understand the amyloidogenesis.

Impact of Turn Propensity on the Folding Rates of Z34C Protein: Implications for the Folding of Helix-Turn-Helix Motif.

The rate-limiting step for the folding of the helix-turn-helix (HTH) protein, Z34C, involves ß-turn region 20DPNL23. This reverse turn has been observed to be part of the transition state in the folding process for Z34C, influencing its folding rates. Molecular dynamics simulations were performed on this turn peptide and its two mutants, D20A and P21A, to study turn formation using GROMOS54A7 force field. We find that this region has a turn propensity of its own, and the highest turn propensity is observed for the wild-type, which correlates well with available experimental results. We also find that a slight unfavorable change in ΔG turn folding causes a drastic change in the folding rates of HTH motif and a mechanistic interpretation is given. Implications of these observations for the folding of the HTH protein Z34C are discussed.

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of ß-turns in peptide immunogenicity.

Earlier immunological experiments with a synthetic 36-residue peptide (75-110) from Influenza hemagglutinin have been shown to elicit anti-peptide antibodies (Ab) which could cross-react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide ((98)YPYDVPDYASLRS(110)) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS-AA/L all-atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (ß-turn formed by "DYAS" sequence) with greater preference at 310 K while, it samples a native-like conformation with relatively greater propensity at 293 K. The sequence "DYAS" samples ß-turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the ß-turn involving "DYAS" sequence and in addition it also samples a ß-turn formed by the sequence "YPYD" at its N-terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag-Ab recognition are discussed.

Environmental polarity induces conformational transitions in a helical peptide sequence from bacteriophage T4 lysozyme and its tandem duplicate: a molecular dynamics simulation study.

Our recent molecular dynamics (MD) simulation of an insertion/duplication mutant 'L20' of bacteriophage T4 lysozyme demonstrated a solvent induced αâ†’ß transition in a loosely held duplicate helical region, while α-helical conformation in the parent region was relatively stabilized by its tertiary interactions with the neighboring residues. The solution NMR of the parent helical sequence, sans its protein context, showed no inherent tendency to adopt a particular secondary structure in pure water but showed α-helical propensity in TFE/water and SDS micelles. In this study we investigate the conformational preference of the 'parent' and 'duplicate' sequences, sans the protein context, in pure water and an apolar TFE/water solution. Apolar TFE/water solution is a model for non-polar protein context. We performed MD simulations of the two peptides, in explicit water and 80% (v/v) TFE/water, using GROMOS 53a6 force field, at 300 K and 1 bar (under NPT conditions). We show that in TFE/water mixture, salt bridges are stabilized by apolar TFE molecules and main chain-main chain hydrogen bonds promote the α-helical conformation, particularly in the duplicate peptide. Solvent exposure, in pure water, resulted in an αâ†’ß transition to form a triple stranded ß-sheet structure in the 'duplicate' sequence, with a rare psi-loop topology, while a mixture of turn/bend conformations were adopted by the 'parent' sequence. Thus the differences in conformational preference of the parent and duplicate sequence sans protein context, in pure water and TFE/water, implicate the importance of the environment polarity in dictating the peptide conformation. Mechanism of folding of the observed psi-loop in the duplicate sequence gives insights into folding of this rare ß-sheet topology.

Energetics of ß-turn formation in a mutant peptide YPGDV from influenza hemagglutinin: an MD simulation study.

Reverse turns play an important role in protein folding, molecular recognition and in eliciting immune response. While sequence determinants of reverse turns are known, not much is known about their energetics. In this paper we have investigated the thermodynamics of a reverse turn sequence YPGDV, an experimentally well characterized turn sequence, using molecular dynamics simulations performed over a range of temperatures from 280-360 K using GROMACS 4.0.4 software and all atom OPLS-AA/L force field. The change in folding free energy (ΔAfolding) for the ß-turn formation in YPGDV peptide shows a linear relationship with temperature. We find that the entropy change (ΔSfolding) for the ß-turn formation is close to zero and the internal energy change (ΔUfolding) is a modest -3.8 kJ mol(-1). These thermodynamic quantities are interpreted in terms of intra-molecular (intra-peptide) and inter-molecular (peptide-solvent) hydrogen bonding interactions. Implications for protein folding and peptide immunogenicity are discussed.

Molecular dynamics simulations of certain mutant peptide models from staphylococcal nuclease reveal that initial hydrophobic collapse associated with turn propensity drives ß-hairpin folding.

An important nucleation event during the folding of staphylococcal nuclease involves the formation of a ß-hairpin by the sequence (21) DTVKLMYKGQPMTFR(35) . Earlier studies show that the turn sequence 'YKGQP' has an important role in the folding of this ß-hairpin. To understand the active or passive nature of the turn sequence 'YKGQP' in the folding of the aforementioned ß-hairpin sequence, we studied glycine mutant peptides Ac-(2) DTVKLMYGGQPMTFR(16) -NMe (K9G:15), Ac-(2) DTVKLMYKGGPMTFR(16) -NMe (Q11G:15), Ac-(2) DTVKLMYGGGPMTFR(16) -NMe (K9G/Q11G:15), and Ac-(2) DTVKLMGGGGGMTFR(16) -NMe (penta-G:15) by using molecular dynamics simulations, starting with two different unfolded states, polyproline II and extended conformational forms. Further, 5mer mutant turn peptides Ac-(2) YGGQP(6) -NMe (K3G:5), Ac-(2) YKGGP(6) -NMe (Q5G:5), Ac-(2) YGGGP(6) -NMe (K3G/Q5G:5), and Ac-(2) GGGGG(6) -NMe (penta-G:5) were also studied individually. Our results show that an initial hydrophobic collapse and loop closure occurs in all 15mer mutants, but only K9G:15 mutant forms a stable native-like ß-hairpin. In the other 15mer mutants, the hydrophobic collapsed state would not proceed to ß-hairpin formation. Of the different simulations performed for the penta-G:15 mutant, in only one simulation a nonnative ß-hairpin conformation is sampled with highly flexible loop region ((8) GGGGG(12) ), which has no specific conformational preference as a 5mer. While the sequence 'YGGQP' in the K3G:5 simulation shows relatively higher ß-turn propensity, the presence of this sequence in K9G:15 peptide seems to be driving the ß-hairpin formation. Thus, these results seem to suggest that for the formation of a stable ß-hairpin, the initial hydrophobic collapse is to be assisted by a turn propensity. Initial hydrophobic collapse alone is not sufficient to guide ß-hairpin formation.

Molecular dynamics study of an insertion/duplication mutant of bacteriophage T4 lysozyme reveals the nature of αâ†’ß transition in full protein context.

An αâ†’ß transition underlies the first step of disease causing amyloidogenesis in many proteins. In view of this, many studies have been carried out using peptide models to characterize these secondary structural transitions. In this paper we show that an insertion/duplication mutant 'L20' of bacteriophage T4 lysozyme (M. Sagermann, W. A. Baase and B. W. Matthews, Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 6078) displays an αâ†’ß transition. We performed molecular dynamics (MD) simulation of L20, using the GROMACS package of programs and united atom GROMOS 53a6 force field for a time period of 600 ns at 300 K, in explicit water. Our MD simulation demonstrated that the transition occurs in a duplicated α-helical region inserted tandemly at the N-terminus of the 'parent' helix. We show that a C-terminal ß-sheet anchors the parent helix while the loosely held N-terminal loop in the duplicate region is vulnerable to solvent attack and thus undergoes an αâ†’ß transition. Main chain-solvent interactions were seen to stabilize the observed ß-structure. Thus L20 serves as a good protein model for characterization of αâ†’ß transition in a full length protein.

The role of loop closure propensity in the refolding of Rop protein probed by molecular dynamics simulations.

Rop protein is a homo-dimer of helix-turn-helix and has relatively slow folding and unfolding rates compared to other dimeric proteins of similar size. Fluorescence studies cited in literature suggest that mutation of turn residues D30-A31 to G30-G31 (Gly2) increases its folding and unfolding rates considerably. A further increase in number of glycines in the turn region results in decrease of folding rates compared to Gly2 mutant. To understand the effect of glycine mutation on folding/unfolding rates of Rop and the conformational nature of turn region involved in formation of early folding species, we performed molecular dynamics simulations of turn peptides, ²5KLNELDADEQ³4 (DA peptide), ²5KLNELGGDEQ³4 (G2 peptide), ²5KLNELGGGDEQ³5 (G3 peptide) and ²5KLNELGGGEQ³4 (G3(') peptide) from Rop at 300 K. Further Wt-Rop and mutant G2-Rop monomers and dimers were also studied separately by molecular dynamics simulations. Our results show that glycine based peptides (G(n) peptides) have a higher loop closure propensity compared to DA. Comparison of monomeric and dimeric Rop simulations suggests that dimeric Rop necessarily requires α(L) conformation to be sampled at D30/G30 position in the turn region. Since glycine (at position 30) can readily adopt α(L) conformation, G(n) loop plays a dual role in both facilitating loop closure as well as facilitating reorganization/packing of helices required for structural adjustment during dimer formation in the folding of Rop. Based on our simulation results and available literature, we suggest a tentative kinetic model for Rop folding which allows us to estimate the contribution of loop closure propensity to the overall folding rates.

Molecular simulation and docking studies of Gal1p and Gal3p proteins in the presence and absence of ligands ATP and galactose: implication for transcriptional activation of GAL genes.

The Gal4p mediated transcriptional activation of GAL genes requires the interaction between Gal3p bound with ATP and galactose and Gal80p. Though numerous studies suggest that galactose and ATP activate Gal3p/Gal1p interaction with Gal80p, neither the mechanism of activation nor the interacting surface that binds to Gal80p is well understood. In this study we investigated the dynamics of Gal3p and Gal1p in the presence and absence of ligands ATP and galactose to understand the role played by dynamics in the function of these proteins through molecular dynamics simulation and protein-protein docking studies. We performed simulations totaling to 510 ns on both Gal1p and Gal3p proteins in the presence and absence of ligands ATP and galactose. We find that, while binding of ligands ATP and galactose to Gal3p/Gal1p do not affect the global conformation of proteins, some local conformational changes around upper-lip helix including insertion domain are observed. We observed that only in the presence of ATP and galactose, Gal3p displays opening and closing motion between the two domains. And because of this motion, a binding interface, which is largely hydrophobic, opens up on the surface of Gal3p and this surface can bind to Gal80p. From our simulation studies we infer probable docking sites for Gal80p on Gal3p/Gal1p, which were further ascertained by the docking of Gal80p on to ligand bound Gal1p and Gal3p proteins, and the residues at the interface between Gal3p and Gal80p are identified. Our results correlate quite well with the existing body of literature on functional and dynamical aspects of Gal1p and Gal3p proteins.

For the sequence YKGQ, the turn and extended conformational forms are separated by small barriers and the turn propensity persists even at high temperatures: implications for protein folding.

The folding of the sequence (21)DTVKLMYKGQPMTFR(35) from staphylococcal nuclease into a ß-hairpin, nucleated by the turn region YKGQP, is known to be an early folding event. With YKGQ being the shortest sequence for a ß-turn model and in view of its importance to the folding of staphylococcal nuclease, we investigated the thermodynamics of turn formation at a range of temperatures from 280 to 380 K, with a regular interval of 10 K. Eleven independent molecular dynamics simulations (under NPT conditions) were performed using the GROMACS package of programs and the OPLS-AA/L all-atom force field, each for a time period of 1 µs. Turn formation is supported by enthalpy at lower temperatures, while entropy supports it at higher temperatures. There are modest free energy barriers between turn and extended conformational ensembles. The turn propensity persists even at elevated temperatures. The role of proline in driving the turn formation has been re-examined, and it is inferred that the absence of proline does not affect turn propensity.

A shorter peptide model from staphylococcal nuclease for the folding-unfolding equilibrium of a beta-hairpin shows that unfolded state has significant contribution from compact conformational states.

It is important to understand the conformational features of the unfolded state in equilibrium with folded state under physiological conditions. In this paper, we consider a short peptide model LMYKGQPM from staphylococcal nuclease to model the conformational equilibrium between a hairpin conformation and its unfolded state using molecular dynamics simulation under NVT conditions at 300K using GROMOS96 force field. The free energy landscape has overall funnel-like shape with hairpin conformations sampling the minima. The "unfolded" state has a higher free energy of approximately 12kJ/mol with respect to native hairpin minimum and occupies a plateau region. We find that the unfolded state has significant contributions from compact conformations. Many of these conformations have hairpin-like topology. Further, these compact conformational forms are stabilized by hydrophobic interactions. Conversion between native and non-native hairpins occurs via unfolded states. Frequent conversions between folded and unfolded hairpins are observed with single exponential kinetics. We compare our results with the emerging picture of unfolded state from both experimental and theoretical studies.

Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease.

A small tripeptide AFA undergoes two state cooperative conformational transitions: implications for conformational biases in unfolded states.

It is important to understand the conformational biases that are present in unfolded states to understand protein folding. In this context, it is surprising that even a short tripeptide like AFA samples folded/ordered conformation as demonstrated recently by NMR experiments of the peptide in aqueous solution at 280 K. In this paper, we present molecular dynamics simulation of the peptide in explicit water using OPLS-AA/L all-atom force field. The results are in overall agreement with NMR results and provide some further insights. The peptide samples turn and extended conformational forms corresponding to minima in free energy landscape. Frequent transitions between the minima are observed due to modest free energy barriers. The turn conformation seems to be stabilized by hydrophobic interactions and possibly by bridging water molecules between backbone donors and acceptors. Thus the peptide does not sample conformations randomly, but samples well defined conformations. The peptide served as a model for folding-unfolding equilibrium in the context of peptide folding. Further, implications for drug design are also discussed.

The sequence TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase displays structural ambivalence and interconverts between alpha-helical and beta-hairpin conformations mediated by collapsed conformational states.

The peptide TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase adopts a helical conformation in the crystal structure and is a site for two hydrated helical segments, which are thought to be helical folding intermediates. Overlapping sequences of four to five residues from the peptide, sample both helical and strand conformations in known protein structures, which are dissimilar to glyceraldehyde-3-phosphate dehydrogenase suggesting that the peptide may have a structural ambivalence. Molecular dynamics simulations of the peptide sequence performed for a total simulation time of 1.2 micros, starting from the various initial conformations using GROMOS96 force field under NVT conditions, show that the peptide samples a large number of conformational forms with transitions from alpha-helix to beta-hairpin and vice versa. The peptide, therefore, displays a structural ambivalence. The mechanism from alpha-helix to beta-hairpin transition and vice versa reveals that the compact bends and turns conformational forms mediate such conformational transitions. These compact structures including helices and hairpins have similar hydrophobic radius of gyration (Rgh) values suggesting that similar hydrophobic interactions govern these conformational forms. The distribution of conformational energies is Gaussian with helix sampling lowest energy followed by the hairpins and coil. The lowest potential energy of the full helix may enable the peptide to take up helical conformation in the crystal structure of the glyceraldehyde-3-phosphate dehydrogenase, even though the peptide has a preference for hairpin too. The relevance of folding and unfolding events observed in our simulations to hydrophobic collapse model of protein folding are discussed.

Beta-hairpins with native-like and non-native hydrogen bonding patterns could form during the refolding of staphylococcal nuclease.

Refolding of staphylococcal nuclease has been studied recently by hydrogen-deuterium exchange and NMR spectroscopy. These studies infer that beta-hairpin formed by strand 2 and strand 3 connected by reverse turn forms early during the refolding of nuclease. Typically, hydrogen-deuterium exchange NMR techniques are usually carried out on a time scale of milliseconds whereas beta-hairpins are known to fold on a much shorter time scale. It follows that in the experiments, the hydrogen-deuterium exchange protection patterns could be arising from a significant population of fully formed hairpins. In order to demonstrate it is the fully formed hairpins which gives rise to the hydrogen-deuterium exchange protection patterns, we have considered molecular dynamics simulation of the peptide (21)DTVKLMYKGQPMTFR(35) from staphylococcal nuclease corresponding to the beta-hairpin region, using GROMOS96 force field under NVT conditions. Starting from unfolded conformational states, the peptide folds into hairpin conformations with native-like and non-native hydrogen bonding patterns. Subsequent to folding, equilibrium conditions prevail. The computed protection factors and atom depth values, at equilibrium, of the various amide protons agree qualitatively with experimental observations. A collection of molecules following the trajectories observed in the simulations can account for experimental observations. These simulations provide a molecular picture of the formed hairpins and their conformational features during the refolding experiments on nuclease, monitored by hydrogen-deuterium exchange.

Insights into the role of the aromatic residue in galactose-binding sites: MP2/6-311G++** study on galactose- and glucose-aromatic residue analogue complexes.

The presence of an aromatic residue (Trp, Phe, Tyr) facing the nonpolar face of galactose is a common feature of galactose-specific lectins. The interactions such as those between the C-H groups of galactose and the pi-electron cloud of aromatic residues have been characterized as weak hydrogen bonds between soft acids and soft bases, largely governed by dispersive and charge transfer interactions. An analysis of the binding sites of several galactose-specific lectins revealed that the spatial position-orientation of galactose relative to the binding site aromatic residue varies substantially. The effect of variations in position-orientations of galactose on the interaction energies of galactose-aromatic residue complexes has not been determined so far. In view of this, MP2/6-311G++** calculations were performed on galactose- and glucose-aromatic residue analogue complexes in eight position-orientations. The results show that the strength of the C-H...pi interactions in galactose-aromatic residue complexes is comparable to that of a hydrogen bond. Rather than the type of aromatic residue, the position-orientation of the saccharide appears to be more critical in determining the strength of their interactions. Earlier studies have found the binding site aromatic residue to be critical, but its role was not clear. This study shows that the aromatic residue is important for discriminating galactose from glucose, in addition to its contribution to binding energy.