HINT 2.30 Manual: Chapter 5I

Usage and Strategy

Objectives

This chapter describes the basic steps you should follow to use the insightII version of Hint. It contains two sections related to the application of Hint and an overview of the Hint strategy. Following are several specific tutorials that cover most of the commonly used features of Hint.

After studying the descriptive material in the Hint Strategy section and performing the later tutorials you should comfortable with the following operations of Hint:

Partitioning both small molecules and biological macromolecules.
Tabulating InterMolecular and IntraMolecular interactions.
Mapping hydropathic surfaces and interactions.
Using SMILES code to create a small molecule object.

Methodology

Methodology for Hint is presented in two sections.

The first section outlines the basic steps involved in a Hint calculation. Commands provided in the Hint module are discussed in general terms in the order in which they are used in a typical calculation. Also included are suggestions about when certain parameter values should or should not be used. For a more detailed discussion of each command, refer to Chapter 4I of this manual.

The second section presents specific strategies used to solve some typical problems using Hint. Much of this information is background material for the Hint tutorials presented below, so it is recommended that you read this section before starting the tutorial lessons.

Basic Steps for a Hint Calculation

To summarize the basic steps involved in the Hint calculation, the process consists of five steps:

Create and modify the input molecule(s).
Partition the molecule(s).
Set up calculation parameters.
Run the calculation.
Analyze the results.

Step 1: Defining the Molecule(s)

A Hint calculation can be performed on any molecule or assembly of molecules that exists in InsightII. (Some Hint calculations are run on two molecules simultaneously.) Molecules can be read from files using the Get Molecule command, created using the Builder module, or may be the result of work performed in another application module such as Discover. Note that Hint does not modify the molecule(s) in any way. Therefore, any desired modifications, such as adding hydrogens to a molecule read from a PDB file, must be done prior to starting the Hint calculation. As Hint relies on the CVFF atom potential types for hydrophobic atom constant assignment, these must be properly set for most molecules before initiation of the calculation. In the sections below, the molecule(s) or assembl(ies) upon which the calculation is performed are referred to as the input molecule(s).

Step 2: Partitioning the Molecule(s)

The key parameter in Hint calculations is the hydrophobic atom constant which must be assigned for each atom in each molecule. This step is termed partitioning as the hydrophobic atom constants are derived from experimental solvent partition experiments. There are two approaches to partitioning employed by Hint. The first method, Calculate, directly utilizes the atom potential types (CVFF) and connection matrix of the molecule to assign hydrophobic atom constants and to calculate a logP (partition coefficient for water/octanol) which is the sum of the hydrophobic atom constants. This is the method of choice for small molecules. For macromolecules composed of distinct monomer units (especially proteins where the monomers are amino acid residues) the Dictionary method is recommended for consistency and reproducibility. Here, Hint assigns the hydrophobic atom constants based on the monomer and atom names. The LogP for the macromolecule is reported by Hint, but this result is only of tangential interest or validity. In either case, Hydrogen_Treatment must be specified. This parameter describes how the Partitioning will treat hydrogens; there are three options: United_Atom (using an approach the treats all hydrogens implicitly as part of their parent heavy atom), Polar_Only (using an approach that treats (only) polar hydrogens explicitly), and Retain_All. Note that the selection of this parameter has implications in following interaction calculations. For example, hydrogen bonding will be incorrectly modeled by Hint if the United_Atom option is chosen. In general the Polar_Only option is best for most applications. The Retain_All option appears to "dilute" the hydrophobic density, but treats aromatic hydrogens as potential hydrogen bond donors.

Calculate Method for Partitioning

If the Calculate method is chosen, one other parameter must be specified: the Polar Proximity. When two or more polar groups are in "in proximity" within a molecule, their effects are cumulatively diminished -- this is termed the polar proximity effect and is dealt with by the Leo method of LogP estimation and by Hint as corrective factors to the hydrophobic atom constants. Hint offers three options for Polar Proximity: Off, Via_Bond, and Through_Space. The Via_Bond method is most compatible with the Leo system, and is the option recommended. Through_Space may be appropriate for some larger molecules that have significant intramolecular non-covalent interactions between polar groups. It uses a through-space distance function to estimate the Polar Proximity effect.

Dictionary Method for Partitioning

The Dictionary method of partitioning requires the selection of an additional parameter to specify the Solvent_Condition for the molecule. The Hint partition dictionary cross references each monomer type and its included atom by three solvent conditions: Acid, Neutral, and Base. For amino acids these conditions refer to fully protonated (both acids and bases), protonated bases and ionized acids, and fully ionized (both acids and bases). A fourth option (Inferred) uses the actual protonation state of the amino acid residue to select the appropriate solvent condition and Hint parameters. This option is to be preferred if the hydrogens of the input molecule (protein) have been very accurately modeled. Missing or superfluous hydrogens will be reflected in an incorrect assignment of the Hint parameters for the monomer, however.

Step 3: Setting up the Calculation Parameters

There are several parameters that can be optimized for different Hint runs. These are generally available through the Setup pulldown in one of six commands that define these parameters. The Setup HINT DistFunct command must be defined for all Hint calculations to control the mathematical behavior of the Hint distance calculation. For any Hint grid calculations, the Grid Setup command must be executed to define the region. The other commands in the Setup pulldown are specific to the type of Hint calculation desired, and the appropriate Setup command must be executed before the actual Hint run.

Defining the Hint Distance Function

The Hint distance function represents the mathematical equation of the distance behavior for interacting atoms in the Hint model. Two terms can be controlled representing the hydrophobic distance effect and the steric distance effect. The former term is not experimentally well understood and is coded in the Hint model as either an exponential or 1/rn. In general the exponential function is more empirically realistic and is recommended for most calculations. The steric term in the overall Hint distance function is simply the Lennard-Jones function representing the dispersion attraction/ repulsion between atoms at or near van der Waals distance from each other. Use of the steric term is optional, and for some grid calculations (e.g., Grid_Molecule and Grid_Complement) the steric term should be off. The Steric/Hydro Scaler parameter balances the contributions of the hydrophobic and steric terms. If using the exponential hydrophobic term, a scaling factor of 50.0 generates an overall interaction that is about 2/3 hydropathic and 1/3 steric between two attractive atoms that are at optimum distance for interaction. The primary contribution of the steric term to the Hint model, however, is to penalize atom-atom contacts that are too close. Another part of the Hint Distance Function is the Direction Vectors which dictate the "shape" of the Distance function. The default condition, None, treats all atoms as being Spherical. The Bond Axes option optimizes the Hint Distance Function for interactions along the direction of molecular bonds while the Hybridizd/Lone Pair applies implied lone pair and pi orbitals (where appropriate) and optimizes the Hint distance function for interactions along these vector axes.

Other parameters that indirectly affect the distance function are CutOff Radius and Van der Waals Limit. The CutOff Radius is a range cutoff that serves to reduce computation time by restricting the Hint calculation to grid points or interactions within range of the atom of interest. Values in the 6-8 Angstrom range are typically used. The Van der Waals Limit parameter is a multiplier for the atomic van der Waals radii to "tighten" or "loosen" the spectrum of acceptable contacts. In theoretical terms, adjustment of the Van der Waals Limit shifts the bottom of the van der Waals interaction potential well. By increasing Van der Waals Limit to 1.10, more atom-atom contacts will be recorded by Hint as being repulsive by virtue of the Hint steric term. Likewise, by reducing Van der Waals Limit to 0.90, some interactions that would have been sterically repulsive would be "allowed". This parameter is included for an additional flexibility in balancing the contributions of the hydropathic and steric terms of the Hint calculation.

Defining the Grid Region

An important part of most Hint calculations involves mapping the molecule or interacting molecules onto a three-dimensional grid. The Grid command defines and displays this grid. There are three basic quantities that must be specified: the Grid Center, Grid Size, and Grid Resolution.

The most common method used to define the grid involves specifying the Molecule_ Region to be placed in the center of the grid, along with a Border_Space to surround this region. A special case arises for InterMolecular interaction maps where the best description of the region may be defined in terms of the Interfacial_Region (or overlap) and Border_Space between two molecules. Alternatively the Grid Center may be specified as any arbitrary coordinate value and the total extent of the grid in the longest dimension may be used to specifiy the Grid Size.

In contrast to DelPhi or other electrostatic mapping programs, there are no "boundary effects" in the Hint calculation. There is no contribution from bulk dielectric. Thus, the selected border space must only satisfy your desire for a complete map. As the exponential distance function most often used in Hint decays much more rapidly than a 1/r2 function, border space in the vicinity of 4-5 Angstrom is usually sufficient.

The only quantity left to specify is the Grid Resolution. To allow flexibility, you may alternatively specify the resolution directly with the Angstroms/Grid Pt parameter, or indirectly by specifying the number of grid points in the longest linear dimension (Max Points on Side) or specifying each axis individually with the Points_Per_ Axis option. Note that even if the Angstroms/Grid Pt parameter is not the chosen method of specifying the grid resolution, the spacing between grid points in all dimensions is consistent, and is based on the Total Grid Extent in the longest dimension divided by the Max Points on Side or Points_Per_Axis for the axis of longest dimension. It is recommended that a grid resolution of 1.0 Angstroms/Grid Pt or less be chosen for the construction of Hint maps due to the rapid decay of the Hint distance function.

By default the Grid command displays the outer edges of the grid to aid in visualization of the enclosed region. The View_Grid_Box parameter controls this display.

It is also possible to adjust the grid center so that the real space origin (0,0,0) would lie exactly on a grid point if the grid were extended in the appropriate direction. The Origin_On_Grid_Pt parameter controls this option, which is included for compatability with other graphics display programs, such as Frodo.

Specifying Grid Map Sub-Types

There are Grid Map Sub-Types that may be specified during the setup of a HintGrid calculation. For the Grid_Molecule and Grid_Complement commands the Grid_Types are Hydrophobic/Polar and Acid/Base. The former map describes hydrophobic portions of the hydropathic field as positive grid points and polar (hydrophilic) portions of the field as negative grid points. The second map, Acid/Base, plots acidic polar regions as positive and basic polar regions as negative (hydrophobic regions are zero). Note that Hint provides three levels of AcidBase Definition models to aid in description of the three-dimensional hydropathic environment of a Molecule or it's Complement. The first level, Coulombic, uses only the formal charge of the atom to categorize acid (positive) or base (negative) properties. The second and third levels are based on the Bronsted and Lewis models, respectively. This option provides additional flexibility in Hint calculations. The default and recommended model is the Lewis acid/base definition as it is the most comprehensive because it recognizes some acid/base interactions not relevant to the Bronsted definition.

For setting up Grid_Interaction maps the Interaction_Select parameter provides an option to split interactions involving Hydrophobic groups/atoms from those that are purely Polar in nature. The All option includes both. The most informative maps are obtained by performing dual calculations (Hydrophobic and Polar) on the same InterMolecular or IntraMolecular interaction.

Defining Table Extents

For HintTable calculations the Table Extents must be set. The Hint interaction calculation includes all atom-atom pairs, but it is usually desirable to limit the scope of the interactions written to the Interaction Table file. You can control the Table Extents with two parameters, Table Radius and Table Value. Table Radius limits the table to atom-atom interactions that are within the specified value. Table Value further refines the Table Extents by restricting the table to interactions having |b_ij| larger than the specified value. Clearly, an InterMolecular HintTable calculation involving two large molecules (e.g., proteins) can have more than 10⁶ specific atom-atom interactions -- an uninterpretable quantity of data. Table Radius of 5-7 and Table Value of 2-10 are customarily chosen and typically yield 10² - 10³ tabulated interactions. The thired Table Extents parameter, Table Resolution, controls the reported significant figures in the table. A further refinement of the Table content is offered by the Proton Suppress parameter. If this parameter is on, protons are not explicitly listed in the HintTable. However the interaction information from the protons is combined with their associated heavy atoms in the table entries. If the Proton Suppress parameter is off, protons in the molecule(s) will be listed as distinct atoms in the table. In either case, the user can control the definition of hydrogen bonds with H- Bnd Distance X-Y if Proton Suppress is on and H-Bnd Distance X-H if Proton Suppress is off.

Using insightII Spreadsheets

Output File Specification

Three types of files are generated by Hint, depending on the specific calculation being performed. Any Partition operation can produce a Partition File (*.par). These are of some interest for archival purposes, but it is generally not necessary to retain these files. HintGrid calculations produce grid files (*.grd) and HintTable calculations produce table files (*.tbl).
The hydropathic maps from HintGrid calculations are generally the most important and useful Hint output files. These contain either the value of the hydropathic density (for Molecule and Complement grids) or the Hint Interaction density at every grid point. Interpretation of the Interaction maps can often be made easier by producing a HintTable hardcopy for the same interaction calculation. This is an ASCII output file that, referenced by atom, lists the specific implicated interactions.

Step 4: Performing Hint Calculations
After all the Setup parameters are specified, you are ready to run the Hint calculation using the commands in either the HintGrid or HintTable pulldown.
Molecule HintGrid
This command will generate a hydropathic map of the input molecule using the grid dimensions selected in Grid Setup and other options selected in . The command prompts you to select the Molecule or other set of atoms to be used in the calculation. This calculation can be performed in one of three ways: Interactive, Background or through a Command_File. If Background or Command_ File are chosen, Grid Filename, Job Name, and Comment parameters are prompted to define the run environment. Interactive causes the calculation to be performed immediately, with loss of InsightII function until the calculation is completed. For small molecules or regions the Molecule calculation takes on the order of a few seconds, such that choosing the Interactive option is not an inconvenience.

IntraMolecular HintGrid
This command will generate an intramolecular hydropathic interaction map of the input molecule using the grid dimensions selected in Grid Setup and other options selected in Grid_Interactions Setup. The command prompts you to select the Molecule or other set of atoms to be used in the calculation. This calculation can be performed in one of three ways: Interactive, Background or through a Command_File. If Background or Command_File are chosen, Grid Filename, Job Name, and Comment parameters are prompted to define the run environment. Interactive causes the calculation to be performed immediately, with loss of InsightII function until the calculation is completed. For large molecules such as proteins the IntraMolecular calculation can take on the order of several hours, such that choosing the Interactive option will rarely be advisable.

InterMolecular HintGrid
This command will generate an intermolecular hydropathic interaction map of the two input molecules using the grid dimensions selected in Grid Setup and other options selected in Grid_Molecule Setup. The command prompts you to select two Molecules or other sets of atoms to be used in the calculation. This calculation can be performed in one of three ways: Interactive, Background or through a Command_File. If Background or Command_File are chosen, Grid Filename, Job Name, and Comment parameters are prompted to define the run environment. Interactive causes the calculation to be performed immediately, with loss of InsightII function until the calculation is completed. For large molecules such as proteins the InterMolecular calculation can take on the order of several hours, such that choosing the Interactive option will rarely be advisable.

Complementary HintGrid
This command will generate a hydropathic map of the unknown complementary species to the input molecule using the grid dimensions selected in Grid Setup and other options selected in Grid_Complement Setup. The command prompts you to select the Molecule or other set of atoms to be used in the calculation. This calculation can be performed in one of three ways: Interactive, Background or through a Command_File. If Background or Command_File are chosen, Grid Filename, Job Name, and Comment parameters are prompted to define the run environment. Interactive causes the calculation to be performed immediately, with loss of InsightII function until the calculation is completed. For small molecules or regions the Complementary calculation takes on the order of a minute or so, such that choosing the Interactive option may not be an inconvenience.

IntraMolecular HintTable
This command will generate an ASCII table of the atom-atom interactions within the input molecule using the options selected in HintTable Setup. The command prompts you to select the Molecule or other set of atoms to be used in the calculation. This calculation can be performed in one of three ways: Interactive, Background or through a Command_File. If Background or Command_File are chosen, Grid Filename, Job Name, and Comment parameters are prompted to define the run environment. Interactive causes the calculation to be performed immediately, with loss of InsightII function until the calculation is completed. This calculation generally takes on the order of a few seconds to a minute or two, such that choosing the Interactive option may not be an inconvenience.

InterMolecular HintTable
This command will generate an ASCII table of the atom-atom interactions between two input molecules using the options selected in HintTable Setup. The command prompts you to select two Molecules or other sets of atoms to be used in the calculation. This calculation can be performed in one of three ways: Interactive, Background or through a Command_File. If Background or Command_File are chosen, Grid Filename, Job Name, and Comment parameters are prompted to define the run environment. Interactive causes the calculation to be performed immediately, with loss of InsightII function until the calculation is completed. This calculation generally takes on the order of a few seconds to a minute or two, such that choosing the Interactive option may not be an inconvenience.

Step 5: Analyzing the Results
Hint calculation results are analyzed in two ways: graphical and analytical. The graphical analysis is accomplished through grid display maps that can be accessed with commands in the Grid pulldown. By far, the best method for analysis of the analytical HintTable results is by obtaining a printout of the file.

HintTable Output (*.tbl)
The HintTable output file contains an itemized atom-by-atom listing of the specific interactions involved in the calculation, including the relevant parameters, the distance between the interacting atoms, and the Hint MicroInteraction constant. All implicated atoms are fully identified and their hydrophobic atom constants and solvent accessible surface areas are listed. The distance between each pair of interacting atoms is tabulated in two ways -- an Angstrom scale, and percentage of van der Waals radii sum. This latter scale permits an obvious scan for problematic close contacts (i.e., those less than 100.0). The Hint MicroInteraction constant is listed in the next column of the HintTable file. This value represents the Hint quantitation of the relative significance of the atom-atom interaction. Positive values represent interactions that are "good", while negative values represent interactions that are "bad" for the substrate binding, protein folding, or quaternary subunit interaction being modeled. The final column lists a preliminary characterization of the interaction type, i.e., whether hydrophobic, acid-base, acid- acid, base-base, hydrophobic-polar, or hydrogen bonding.

Molecule and Complement HintGrids
The most useful method of analysis for HintGrids is to display them as three-dimensional contours. You can create a contour by using the Contour command in the Grid pulldown. Typical contoured values are on the order of +20 to -40. For a HintGrid map calculated with the Grid_Type set as Hydrophobic/Polar the positive contours represent hydrophobic regions of the molecule or assembly and the negative contours represent the polar or hydrophilic regions of the molecule or assembly. (For Complement HintGrids these are regions of the implied interacting species.) Maps calculated as Acid/Base give further information on the Polar portion of the HintGrid by categorizing regions as Acidic or Basic. If both Grid_Types have been calculated for a molecule or assembly, you can get the best combination of information by contouring the positive portion of the Hydrophobic/Polar map and both the negative and positive portions of the Acid/Base map.

IntraMolecular and InterMolecular HintGrids
IntraMolecular and InterMolecular HintGrids provide unique tools to visualize non-covalent interactions within or between molecules and/or assemblies. You can create a contour by using the Contour command in the Grid pulldown. Typical contoured values for interaction maps are on the order of -400 to +400. The most information can be obtained by calculating (and mapping) the Polar interactions separate from the Hydrophobic interactions. Then, positive contours of the Hydrophobic map represent hydrophobic-hydrophobic interactions; negative contours represent polar-hydrophobic interactions which are generally unfavorable. It is advisable to not over-evaluate the importance of this contribution as this type of interactions is virtually unavoidable in the biological environment. Positive contours of the Polar map represent polar interactions such as acid-base, hydrogen bonding and Coulombic effects; negative contours represent unfavorable polar interactions such as acid-acid, base-base, etc. These latter negative effects may often be reduced by the contribution of water molecules acting as hydrogen bond donors/acceptors or by the protonation or deprotonation of acids and/or bases at the interface.

Typical HINT Scenarios
The preceding section described how to setup and run some Hint calculations without particular regard to the problem being solved. This section describes several common types of interaction calculations for which Hint is appropriate and the strategies used to obtain the desired results. This is not intended as an exhaustive list of the uses of Hint, but rather as an introduction to some underlying principles that can be extended to many other types of applications.

Examination of Hydropathic Field around Molecule
One of the most common uses of Hint is to calculate the hydropathic potential field around a small molecule or macromolecule, and display this result as three-dimensional contours. This provides qualitative information that may be used to explain the interaction of the molecule with other molecules, or suggest a rationale for the observed structure of the molecule (i.e., intramolecular non-covalent forces). The most detailed information would be obtained by parallel runs using the Hydrophobic/Polar and Acid/Base Grid_Types, which can be contoured together to display the hydrophobic, acidic, and basic regions of the molecule. The preferred Hint Distance_ Function for Molecule maps is one using the exponential hydropathic term and no steric term.
After the Molecule HintGrid calculation has completed, you can create the contours using the Contour command of the Grid pulldown.

Prediction of Hydropathic Field for Complementary Species
Hint can be used to predict the Hydropathic Field for a Complementary species. When the structure of a receptor is known it is useful to qualitatively identify the hydropathic structural features of the ideal Complementary molecule (termed the "Key"). With this information it may be possible to construct a molecule with those features, that would be presumed to be the "best" substrate for the receptor site. Likewise, with a known drug, a "Lock" map can be constructed that may reveal the hydropathic structural features of an unknown receptor.
For Grid_Complement calculations, Using the Direction Vector option in the HINT DistFunct setup is advisable as it will enhance the probability of map density being placed in the unoccupied regions of space and optimizes the probability of front-side interactions.

Tabulation/Visualization of InterMolecular Interactions
The best visualization of InterMolecular interactions is obtained by using two Hint runs. One to calculate the Hydrophobic interactions and the second to calculate the Polar interactions. Both runs will be calculated over the same grid space, and can be contoured and displayed simultaneously. Interpretation of the maps is aided by the preparation of a HintTable for the same InterMolecular interaction. This, in essence, will give you a legend to the maps.
The preferred HINT DistFunct for interaction maps is one which includes both the exponential Hydropathic term and the Steric term. We have found a Steric/ Hydro Scaler of 50.0 to be satisfactory in its balance of the two terms. Note that the majority of quantitatively large negative interactions are due to steric violations where the two interacting atoms are too close to each other.

Analysis of a Series of Substrates
If you have a series of molecules known or suspected to bind at the same receptor Hint provides tools for analysis of these data. If the receptor structure is known, you can perform InterMolecular (Binding) HintTable calculations for each substrate with the receptor. The resulting Hint interaction constants may be then correlated with known binding constants for the substrate/receptor binding to construct a calibration curve for the system. Hint has been shown in several cases to order predicted vs. actual binding constants. The next obvious step, once a calibration curve is available, is to predict the binding constants for new molecules. It is important to note the large number of potential uncertainties in this approach. Each atom- atom interaction contributes to the total interaction constant; and each atom-atom microbinding constant is significantly dependent on the distance between the atoms, and by inference, the quality of the molecular model construction.
A second approach to analysis of a series of substrates is one that does not rely on a known receptor structure. That is the LockSmith model. In LockSmith, the series of molecules are analyzed in terms of their three-dimensional hydropathic maps and their measured activities. The key modeling step is construction of an overlap model for the series that represents the presumed relative orientation of the molecules at the (unknown) receptor. Then each molecule is subjected to a HintGrid calculation to obtain the three-dimensional hydropathic structure of the molecules in the set. LockSmith combines these maps by factoring in the molecular biological activities to obtain a single map encoding the activity-weighted hydropathic structural features of the series.
The Activity command of the LockSmith pulldown has tools to convert the Raw_Activity into a Functnalzd_Activity that is appropriate for the LockSmith model creation. After the LockSmith map is calculated (using the Include command), the Make Model command scores each component map to the LockSmith map and produces a best-fit equation relating the scores to the activities. The Predict command fits a new hydropathic map to the model equation to predict biological activity for the new molecule.

Hydropathic Studies of Site-Directed Mutations
Both the Molecule and Interaction HintGrids can be useful tools for investigating the effects of site-directed protein mutations. The Difference command in the HintGrid pulldown will take the difference (subtract or add) between two grids. One strategy would be to compare the Hydropathic maps for "native" and "mutant" Molecules to determine the effect of the mutation on Hydropathic structure. This could reveal, for instance, whether there is an increase in hydrophobicity at the active site due to the mutation.
If the mutation involves structural features implicated in substrate binding, at a subunit interface or in protein folding, difference grids from InterMolecular or IntraMolecular interaction maps may be useful tools to gain a qualitative understanding of the effect of the mutation on the non-covalent forces at the molecular interface.