Tethered-Hopping Model for Protein-DNA Binding and Unbinding Based on Sox2-Oct1-Hoxb1 Ternary Complex Simulations

The sliding and hopping models encapsulate the essential protein-DNA binding process for binary complex formation and dissociation. However, the effects of a cofactor protein on the protein-DNA binding process that leads to the formation of a ternary complex remain largely unknown. Here we investigate the effect of the cofactor Sox2 on the binding and unbinding of Oct1 with the Hoxb1 control element. We simulate the association of Oct1 with Sox2-Hoxb1 using molecular dynamics simulations, and the dissociation of Oct1 from Sox2-Hoxb1 using steered molecular dynamics simulations, in analogy to a hopping event of Oct1. We compare the kinetic and thermodynamic properties of three model complexes (the wild-type and two mutants) in which the Oct1-DNA base-specific interactions or the Sox2-Oct1 protein-protein interactions are largely abolished. We find that Oct1-DNA base-specific interactions contribute significantly to the total interaction energy of the ternary complex, and that nonspecific Oct1-DNA interactions are sufficient for driving the formation of the protein-DNA interface. The Sox2-Oct1 protein-protein binding interface is largely hydrophobic, with remarkable shape complementarity. This interface promotes the formation of the ternary complex and slows the dissociation of Oct1 from its DNA-binding site. We propose a simple two-step reaction model of protein-DNA binding, called the tethered-hopping model, that explains the importance of the cofactor Sox2 and may apply to similar ternary protein-DNA complexes.     

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.     

Molecular Simulations of a Dynamic Protein Complex: Role of Salt-Bridges and Polar Interactions in Configurational Transitions

Ion charge pairs and hydrogen bonds have been extensively studied for their roles in stabilizing protein complexes and in steering the process of protein association. Recently, it has become clear that some protein complexes are dynamic in that they interconvert between several alternate configurations. We have previously characterized one such system: the EphA2:SHIP2 SAM-SAM heterodimer by solution NMR. Here we carried out extensive all-atom molecular-dynamics simulations on a microsecond time-scale starting with different NMR-derived structures for the complex. Transitions are observed between several discernible configurations at average time intervals of 50–100 ns. The domains reorient relative to one another by substantial rotation and a slight shifting of the interfaces. Bifurcated and intermediary salt-bridge and hydrogen-bond interactions play a role in the transitions in a process that can be described as moving along a “monkey-bar”. We notice an increased density of salt bridges near protein interaction surfaces that appear to enable these transitions, also suggesting why the trajectories can become kinetically hindered in regions where fewer of such interactions are possible. In this context, even microsecond molecular-dynamics simulations are not sufficient to sample the energy landscape unless the structures remain close to their experimentally derived low-energy configurations.     

Downhill binding energy surface of the barnase–barstar complex

We have employed biased molecular dynamics simulations in explicit solvent to characterize the one‐dimensional potential of mean force for the dissociation process of the barnase–barstar protein–protein complex. Unbinding of barstar from wild‐type barnase was compared with dissociation from four charge‐deletion mutants of barnase. Interestingly, we find in all cases that unbinding of barnase and barstar is an uphill process on a smooth, tilted energy landscape. The total free energy difference between the dissociated and bound state was similar for wild‐type barnase–barstar and for the R87A mutant of barnase. The values for the three other mutant barnase mutants K27A, R59A, and R83Q were only about half as much. Besides, we have analyzed the conformational dynamics of important residues at the barnase–barstar interface. In the bound state, their conformational fluctuations are reduced relatively to the free state because of the formation of intermolecular contacts. Interestingly, we find that some residues also show decreased mobility at intermediate stages of the unbinding process suggesting that these residues may be involved in the first contacts being formed on binding. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 977–985, 2010.     

The binding process of a nonspecific enzyme with DNA

Protein-DNA recognition of a nonspecific complex is modeled to understand the nature of the transient encounter states. We consider the structural and energetic features and the role of water in the DNA grooves in the process of protein-DNA recognition. Here we have used the nuclease domain of colicin E7 (N-ColE7) from Escherichia coli in complex with a 12-bp DNA duplex as the model system to consider how a protein approaches, encounters, and associates with DNA. Multiscale simulation studies using Brownian dynamics and molecular-dynamics simulations were performed to provide the binding process on multiple length- and timescales. We define the encounter states and identified the spatial and orientational aspects. For the molecular length-scales, we used molecular-dynamics simulations. Several intermediate binding states were found, which have different positions and orientations of protein around DNA including major and minor groove orientations. The results show that the contact number and the hydrated interfacial area are measures that facilitate better understanding of sequence-independent protein-DNA binding landscapes and pathways.     

On the roles of substrate binding and hinge unfolding in conformational changes of adenylate kinase

We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier works of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro¹⁷⁷ on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro¹⁷⁷ hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.     

Molecular Basis for the Dissociation Dynamics of Protein A-Immunoglobulin G1 Complex

Staphylococcus aureus protein A (SpA) is the most popular affinity ligand for immunoglobulin G1 (IgG1). However, the molecular basis for the dissociation dynamics of SpA-IgG1 complex is unclear. Herein, coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field were used to study the dissociation dynamics of the complex. The CG-MD simulations were first verified by the agreement in the structural and interactional properties of SpA and human IgG1 (hIgG1) in the association process between the CG-MD and all-atom MD at different NaCl concentrations. Then, the CG-MD simulation studies focused on the molecular insight into the dissociation dynamics of SpA-hIgG1 complex at pH 3.0. It is found that there are four steps in the dissociation process of the complex. First, there is a slight conformational adjustment of helix II in SpA. This is followed by the phenomena that the electrostatic interactions provided by the three hot spots (Glu143, Arg146 and Lys154) of helix II of SpA break up, leading to the dissociation of helix II from the binding site of hIgG1. Subsequently, breakup of the hydrophobic interactions between helix I (Phe132, Tyr133 and His137) in SpA and hIgG1 occurs, resulting in the disengagement of helix I from its binding site of hIgG1. Finally, the non-specific interactions between SpA and hIgG1 decrease slowly till disappearance, leading to the complete dissociation of the SpA-hIgG1 complex. This work has revealed that CG-MD coupled with the Martini force field is an effective method for studying the dissociation dynamics of protein-protein complex.     

Crystal Structure of the HEAT Domain from the Pre-mRNA Processing Factor Symplekin

The majority of eukaryotic pre-mRNAs are processed by 3′-end cleavage and polyadenylation, although in metazoa the replication-dependent histone mRNAs are processed by 3′-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the ∼ 1160-residue protein Symplekin. Secondary-structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 Å resolution with single-wavelength anomalous dispersion phasing methods. The structure exhibits five canonical HEAT repeats along with an extended 31-amino-acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3′-end processing. Together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process.     

Effects of tryptophan to phenylalanine substitutions on the structure, stability, and enzyme activity of the IIAB(Man) subunit of the mannose transporter of Escherichia coli.

The hydrophilic subunit of the mannose transporter (IIAB(Man)) of Escherichia coli is a homodimer that contains four tryptophans per monomer, three in the N-terminal domain (Trp12, Trp33, and Trp69) and one in the C-terminal domain (Trp182). Single and double Trp-Phe mutants of IIABMan and of the IIA domain were produced. Fluorescence emission studies revealed that Trp33 and Trp12 are the major fluorescence emitters, Trp69 is strongly quenched in the native protein and Trp182 strongly blue shifted, indicative of a hydrophobic environment. Stabilities of the Trp mutants of dimeric IIA(Man) and IIAB(Man) were estimated from midpoints of the GdmHCl-induced unfolding transitions and from the amount of dimers that resisted dissociation by SDS (sodium dodecyl sulfate), respectively. W12F exhibited increased stability, but only 6% of the wild-type phosphotransferase activity, whereas W33F was marginally and W69F significantly destabilized, but fully active. Second site mutations W33F and W69F in the background of the W12F mutation reduced protein stability and suppressed the functional defect of W12F. These results suggest that flexibility is required for the adjustments of protein-protein contacts necessary for the phosphoryltransfer between the phosphorylcarrier protein HPr, IIA(Man), IIB(Man), and the incoming mannose bound to the transmembrane IIC(Man)-IID(Man) complex.     

Protein-protein docking by simulating the process of association subject to biochemical constraints

We present a computational procedure for modeling protein-protein association and predicting the structures of protein-protein complexes. The initial sampling stage is based on an efficient Brownian dynamics algorithm that mimics the physical process of diffusional association. Relevant biochemical data can be directly incorporated as distance constraints at this stage. The docked configurations are then grouped with a hierarchical clustering algorithm into ensembles that represent potential protein-protein encounter complexes. Flexible refinement of selected representative structures is done by molecular dynamics simulation. The protein-protein docking procedure was thoroughly tested on 10 structurally and functionally diverse protein-protein complexes. Starting from X-ray crystal structures of the unbound proteins, in 9 out of 10 cases it yields structures of protein-protein complexes close to those determined experimentally with the percentage of correct contacts >30% and interface backbone RMSD <4 A. Detailed examination of all the docking cases gives insights into important determinants of the performance of the computational approach in modeling protein-protein association and predicting of protein-protein complex structures.     

A combined molecular dynamics and rapid kinetics approach to identify conserved three-dimensional communication networks in elongation factor Tu

Elongation factor (EF) Tu delivers aminoacyl-tRNAs to the actively translating bacterial ribosome in a GTP-hydrolysis-dependent process. Rapid recycling of EF-Tu, catalyzed by EF-Ts, is required for efficient protein synthesis in vivo. Here we report a combined theoretical and experimental approach aimed at identifying three-dimensional communication networks in EF-Tu. As an example, we focus on the mechanistic role of second-shell residue Asp¹⁰⁹. We constructed full-length structural models of EF-Tu from Escherichia coli in the GDP-/GTP-bound state and performed several 10-ns-long molecular-dynamics simulations. During these simulations, the side chain of Asp¹⁰⁹ formed a previously undetected transient hydrogen bond to His²², an invariant residue in the phosphate-binding loop (P-loop). To experimentally validate our molecular-dynamics results and further analyze the role of this hydrogen bond, we determined all rate constants for the multistep reaction between EF-Tu (wild-type and two mutants), EF-Ts, GDP, and GTP using the stopped-flow technique. This mutational analysis revealed that the side chain of Asp¹⁰⁹ is important for acceleration of GDP, but not for GTP dissociation by EF-Ts. The possibility that the Asp¹⁰⁹ side chain has a role in transition-state stabilization and coupling of P-loop movements with rearrangements at the base side of the nucleotide is discussed.     

Interactions Between a Voltage Sensor and a Toxin via Multiscale Simulations

Gating-modifier toxins inhibit voltage-gated ion channels by binding the voltage sensors (VS) and altering the energetics of voltage-dependent gating. These toxins are thought to gain access to the VS via the membrane (i.e., by partitioning from water into the membrane before binding the VS). We used serial multiscale molecular-dynamics (MD) simulations, via a combination of coarse-grained (CG) and atomistic (AT) simulations, to study how the toxin VSTx1, which inhibits the archeabacterial voltage-gated potassium channel KvAP, interacts with an isolated membrane-embedded VS domain. In the CG simulations, VSTx1, which was initially located in water, partitioned into the headgroup/water interface of the lipid bilayer before binding the VS. The CG configurations were used to generate AT representations of the system, which were subjected to AT-MD to further evaluate the stability of the complex and refine the predicted VS/toxin interface. VSTx1 interacted with a binding site on the VS formed by the C-terminus of S1, the S1-S2 linker, and the N-terminus of S4. The predicted VS/toxin interactions are suggestive of toxin-mediated perturbations of the interaction between the VS and the pore domain of Kv channels, and of the membrane. Our simulations support a membrane-access mechanism of inhibition of Kv channels by VS toxins. Overall, the results show that serial multiscale MD simulations may be used to model a two-stage process of protein-bilayer and protein-protein interactions within a membrane.     

Unveiling the Unfolding Pathway of F5F8D Disorder-Associated D81H/V100D Mutant of MCFD2 via Multiple Molecular Dynamics Simulations

Abstract

Combined factor deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in the LMAN1 or MCFD2 genes. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, EF-hand-Ca²⁺ dissociation and assembly of misfolded MCFD2-LMAN1 complex. Here, we have investigated the solution conformations of the MCFD2^(D81H,V100D) protein mutant through extensive molecular dynamics (MD) simulations. The V100D, one of the many MCFD2 mutations known to be associated to F5F8D, is difficult to be reconciled with the pathway model because it is located far from the metal sites and the MCFD2/LMAN1 interface. Consequently, an inspection of all the steps involved in D81H/V100D MCFD2 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. A comparison with parallel studies carried out for the Wild-Type (WT) MCFD2 pointed out that the mutation decreases the affinity of the protein for the Ca²⁺ ion. Multiple explicit solvents MD simulations (50 ns) performed on the two proteins revealed that in the WT protein, stable H-bond network and compact hydrophobic core region are created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. In fact it is shown that the V100D mutation, although located far away the EF-hand domain, may induce subtle modification in the structural core of MCFD2 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway. The native-like hydrophobic cluster formed near the V100 residue in the wild-type protein is disrupted by the negatively charged Asparagine residue. Furthermore, the presence of the D81H mutation in the EF-1 hand domain may also increase the protein unfolding rate and consequently prevent the formation of the MCFD2-LMAN1 complex. The detailed structural insights obtained from our large-scale simulations complement the clinical features and offer useful insights into the mechanism behind MCFD2 protein misfolding.     

The salt dependence of the interferon regulatory factor 1 DNA binding domain binding to DNA reveals ions are localized around protein and DNA

The equilibrium dissociation constant of the DNA binding domain of interferon regulatory factor 1 (IRF1 DBD) for its DNA binding site depends strongly on salt concentration and salt type. These dependencies are consistent with IRF1 DBD binding to DNA, resulting in the release of cations from the DNA and both release of anions from the protein and uptake of a cation by the protein. We demonstrated this by utilizing the fact that the release of fluoride from protein upon complex formation does not contribute to the salt concentration dependence of binding and by studying mutants in which charged residues in IRF1 DBD that form salt bridges with DNA phosphates are changed to alanine. The salt concentration dependencies of the dissociation constants of wild-type IRF1 DBD and the mutants R64A, D73A, K75A, and D73A/K75A were measured in buffer containing NaF, NaCl, or NaBr. The salt concentration and type dependencies of the mutants relative to wild-type IRF1 DBD provide evidence of charge neutralization by solution ions for R64 and by a salt bridge between D73 and K75 in buffer containing chloride or bromide salts. These data also allowed us to determine the number, type, and localization of condensed ions around both IRF1 DBD and its DNA binding site.     

Unveiling the Unfolding Pathway of F5F8D Disorder-Associated D81H/V100D Mutant of MCFD2 via Multiple Molecular Dynamics Simulations

Combined factor deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in the LMAN1 or MCFD2 genes. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, EF-hand-Ca21 dissociation and assembly of misfolded MCFD2-LMAN1 complex. Here, we have investigated the solution conformations of the MCFD2((D81H,V100D)) protein mutant through extensive molecular dynamics (MD) simulations. The V100D, one of the many MCFD2 mutations known to be associated to F5F8D, is difficult to be reconciled with the pathway model because it is located far from the metal sites and the MCFD2/LMAN1 interface. Consequently, an inspection of all the steps involved in D81H/V100D MCFD2 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. A comparison with parallel studies carried out for the Wild-Type (WT) MCFD2 pointed out that the mutation decreases the affinity of the protein for the Ca21 ion. Multiple explicit solvents MD simulations (50_ns) performed on the two proteins revealed that in the WT protein, stable H-bond network and compact hydrophobic core region are created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. In fact it is shown that the V100D mutation, although located far away the EF-hand domain, may induce subtle modification in the structural core of MCFD2 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway. The native-like hydrophobic cluster formed near the V100 residue in the wild-type protein is disrupted by the negatively charged Asparagine residue. Furthermore, the presence of the D81H mutation in the EF-1 hand domain may also increase the protein unfolding rate and consequently prevent the formation of the MCFD2-LMAN1 complex. The detailed structural insights obtained from our large-scale simulations complement the clinical features and offer useful insights into the mechanism behind MCFD2 protein misfolding.     

Photoproducts of bacteriorhodopsin mutants: a molecular dynamics study.

Molecular dynamics simulations of wild-type bacteriorhodopsin (bR) and of its D85N, D85T, D212N, and Y57F mutants have been carried out to investigate possible differences in the photoproducts of these proteins. For each mutant, a series of 50 molecular dynamics simulations of the photoisomerization and subsequent relaxation process were completed. The photoproducts can be classified into four distinct classes: 1) 13-cis retinal, with the retinal N-H+ bond oriented toward Asp-96; 2) 13-cis retinal, with the N-H+ oriented toward Asp-85 and hydrogen-bonded to a water molecule; 3) 13,14-di-cis retinal; 4) all-trans retinal. Simulations of wild-type bR and of its Y57F mutant resulted mainly in class 1 and class 2 products; simulations of D85N, D85T, and D212N mutants resulted almost entirely in class 1 products. The results support the suggestion that only class 2 products initiate a functional pump cycle. The formation of class 1 products for the D85N, D85T, and D212N mutants can explain the reversal of proton pumping under illumination by blue and yellow light.     

Molecular dynamics as a tool to detect protein foldability. A mutant of domain B1 of protein G with non-native secondary structure propensities

The usefulness of molecular dynamics to assess the structural integrity of mutants containing several mutations has been investigated. Our goal was to determine whether molecular dynamics would be able to discriminate mutants of a protein having a close-to-wild-type fold, from those that are not folded under the same conditions. We used as a model the B1 domain of protein G in which we replaced the unique central alpha-helix by the sequence of the second beta-hairpin, which has a strong intrinsic propensity to form this secondary structure in solution. In the resulting protein, one-third of the secondary structure has been replaced by a non-native one. Models of the mutants were built based on the three-dimensional structure of the wild-type GB1 domain. During 2 ns of molecular dynamics simulations on these models, mutants containing up to 10 mutations in the helix retained the native fold, while another mutant with an additional mutation unfolded. This result is in agreement with our circular dichroism and NMR experiments, which indicated that the former mutants fold into a structure similar to the wild-type, as opposed to the latter mutant which is partly unfolded. Additionally, a mutant containing six mutations scattered through the surface of the domain, and which is unfolded, was also detected by the simulation. This study suggests that molecular dynamics calculations could be performed on molecular models of mutants of a protein to evaluate their foldability, prior to a mutagenesis experiment.     

NMR structure of a heterodimeric SAM:SAM complex: characterization and manipulation of EphA2 binding reveal new cellular functions of SHIP2

The sterile alpha motif (SAM) for protein-protein interactions is encountered in over 200 proteins, but the structural basis for its interactions is just becoming clear. Here we solved the structure of the?EphA2-SHIP2 SAM:SAM heterodimeric complex by use of NMR restraints from chemical shift perturbations, NOE and RDC experiments. Specific contacts between the protein surfaces differ significantly from a previous model and other SAM:SAM complexes. Molecular dynamics and docking simulations indicate fluctuations in the complex toward alternate, higher energy conformations. The interface suggests that EphA family members bind to SHIP2 SAM, whereas EphB members may not; correspondingly, we demonstrate binding of EphA1, but not of EphB2, to SHIP2. A variant of EphB2 SAM was designed that binds SHIP2. Functional characterization of a mutant EphA2 compromised in SHIP2 binding reveals two previously unrecognized functions of SHIP2 in suppressing ligand-induced activation of EphA2 and in promoting receptor coordinated chemotactic cell migration.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Theoretical studies of the response of a protein structure to cavity-creating mutations

We have investigated the response of a protein structure to cavity-creating mutations by molecular dynamics (MD) simulations for the wild-type and the five mutants of phage T4 lysozyme. Essential dynamics (ED) analysis and the methods for calculating different components of local interaction energies are used to examine the structural and energetic characteristics associated with the mutations. In agreement with the x-ray results, it is found that the structural changes due to the replacements of a bulky side chain such as Leu or Phe with Ala within the hydrophobic core can be characterized as slight adjustments rather than substantial reorganization of the protein. The relative stability of different mutant structures can be related with the extent of structural readjustments in response to the mutation. The destabilization of the mutant Leu-->Ala proteins relative to the wild-type is closely related with the loss of van der Waals contacts due to the cavity-creating mutations.