1 june 2015dimacs - machine learning in bioinformatics 1 machine learning as applied to structural...

April 18, 2023 DIMACS - Machine Learning in Bioinformatics

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Machine Learning as Applied to Structural Bioinformatics: Results and Challenges

Philip E. Bourne

University of California San Diego

[email protected]


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The Current Situation

• Structure contributes greatly to our understanding of living systems

• We are locked into thinking about structure in specific ways which limits our view– All too often we consider

structure as a static entity

– The view at left is not how another protein or a small molecule ligand sees PKA

• We are still not very good at certain problems …


3

Example Unsolved Problems that Machine Learning Can Address

• Predicting flexibility and disorder in protein structure• Predicting sites of protein-protein and protein-ligand

interaction• Predicting protein function• Defining domain boundaries from sequence• Predicting secondary, tertiary and quaternary

structure• Predicting what will crystallize


4

Example Unsolved Problems that Machine Learning Can Address

• Predicting flexibility and disorder in protein structure• Predicting sites of protein-protein and protein-ligand

interaction• Predicting protein function• Defining domain boundaries from sequence• Predicting secondary, tertiary and quaternary

structure• Predicting what will crystallize

* Will talk about this* Will offer as a challenge


5

The Current Situation: The Potential “Training Set” is Growing Quickly

• High level of redundancy as measured by sequence or structure

• Structure space is clearly very finite, but not clear how much is covered

• Increase in functionally uncharacterized structures

• Complexity is increasing, but still lack complexes

• Structures predominantly 1 and 2 domains

• Lack membrane proteins

• In summary the training set is still not truly representative but structural genomics will improve this situation


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Predicting Functional Flexibility

Jenny Gu

Gu, Gribskov & Bourne PLoS Computational Biology 2006 Early On-line Release


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Spectrum of Protein Order and Disorder

OrderedStructures

DisorderedStructures

If we believe that the 3-dimensional structure of a protein is defined by its 1-dimensional sequence then why not its

flexibility?


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Bridging the Sequence-flexibility Gap

Generalize sequence - flexibility relationship to identify local protein

regions important for allostery


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The Training Dataset

The dataset contains the following qualities:

• Non-redundant sequences– training set with sequences containing ≤ 10% identity.

• With good quality structures– R-factor < 0.30

• At high resolution– Resolution < 2.0 Å.

Total number of proteins in dataset: 1277 sequences


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Obtaining Protein Dynamic Information

Protein structures treated as a 3-D elastic network.

Bahar, I., A.R. Atilgan, and B. Erman

Direct evaluation of thermal fluctuations in proteins using a single-parameter harmonic potential.

Folding & Design, 1997. 2(3): p. 173-181.


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Defining the Target Features

Gaussian Network Model:

• Models protein structure as a 3-D elastic network.

– Each Ca is a node in the network.

– Each node undergoes Gaussian-distributed fluctuations influenced by neighboring interactions within a given cutoff distance. (7Å)

• Decompose protein fluctuation into a summation of different modes.

Bahar, I., A.R. Atilgan, and B. Erman

Direct evaluation of thermal fluctuations in proteins using a single-parameter harmonic potential.

Folding & Design, 1997. 2(3): p. 173-181.


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Side Note: Gaussian Network Model vs Molecular Dynamics

• GNM relatively cause grained

• GNM fast to compute vs MD– Look over larger time scales– Suitable for high throughput


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Functional Flexibility Score

• Utilize correlated movements to help define regional flexibility with functional importance.

Functionally Flexible Score

For each residue:

1. Find Maximum and Minimum Correlation

2. Use to scale normalized fluctuation to determine functional importance

Example: Identifying Functional Flexible Regions (FFR) in HIV Protease

Gu, Gribskov & BournePLoS Comp. Biol.. 2006 Early Release

Correlated modes (yellow)Anti-correlated (blue)

Normalized scores – single chain


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Identifying Regions in Bovine Pancreatic Trypsin Inhibitor and Calmodulin


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How to Represent the Protein Sequence?

• Residues characterized as FFs or not – approx 20% of residues with lengths typically 9+/-11

• The longer the protein the longer the FFR• We use hidden Markov models to represent each

protein sequence in the training dataset.• Hidden Markov models captures evolutionary

information along with the probability of finding one of the 20 amino acids in each position of the sequence.

• Use probability states as input features in the first layer of an architecture containing two SVM layers.


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Architecture of Wiggle

CapturesEvolutionary

Effects

CapturesLocal

Effects(smoothing)

9*29 featuresused for each residue


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Generating Additional Input FeaturesModified Bootstrapping – for Tripeptides – Accounts for Nearest Neighbors Effects

Calculate Z score and P value for each pattern

with respective null models

Sample with replacement44645 times

Pooled Patterns(window size : 3)

Null Model* for FFR Regions

Null Model* for Non-FFR Regions

Sample with replacement199515 times

* Generate 10,000 Null Models


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Architecture of Wiggle

CapturesEvolutionary

Effects

CapturesLocal

Effects(smoothing)

9*29 featuresused for each residue


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Predictors Trained on the Entire Dataset Perform Poorly on Smaller Proteins.

False Positive

False Negative

The characteristics of small proteins are different – eg percent of complexes


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Partition Training Set Based on Sequence Length

• Prediction performance of SVM trained on a partitioned dataset (solid lines) is compared to that was trained on the entire dataset (dashed line).

• Prediction quality improved when dataset is partitioned. Most notably for proteins up to 200 amino acid residues long. Slight improvements observed for proteins longer than 200 residues.

<200 AA Long >200 AA Long


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Performance of Wiggle Predictors

Wiggle

Accuracy: 66.01%

Precision: 37.11%

Recall: 70.49%

Wiggle 200

Accuracy: 76.46%

Precision: 48.99%

Recall: 78.27%


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Case Study: PvuII Endonuclease

FF SCORE

(homodimer for DNA specific cleavage)

Wiggle 200

• Identify known loop for minor grove recognition • Identify hinge residues not previously seen • Important result for mutagenesis studies


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Conclusions for Wiggle

• FFRs can be measured from structure• With some empirical effort these data can be used as

input to an SVM to predict FFRs from sequence alone

• Useful for:– Improving docking studies– Better understand protein function– Engineer more or less stable proteins– ……

Gu, Gribskov & Bourne 2006PLoS Comp. Biol.. 2006 Early Release


25

Exploiting Sequence and Structure Homologs to Identify

Protein-Protein Binding Sites

JoLan Chung

Chung, Wang & Bourne 2006 Proteins: Structure, Function and Bioinformatics, 62(3)

630-640


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Methods to Identify Protein-protein Binding Sites

• Docking• Threading and homology modeling• Evolutionary tracing• Correlated mutations• Properties of patches• Hydrophobicity• Neural networks and support vector machines

(SVM)


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• None of the above methods consider the residues which are spatially conserved on the surfaces of structure homologs

• These residues are reported to correspond to the energy hot spots on protein interfaces and can be derived from multiple structure alignments

Structurally Conserved Surface Residues?


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Method: Incorporate Structural Conservation to Predict the Interface Residue Using SVM

Support vector machine

Sequence + structure information

Binding site location


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Derive the Structurally Conserved Residues

• The structural conservation scores were derived from multiple structural alignments and weighted by the normalized B-factors to consider the structure flexibility that will result in a bad alignment (could use FFRs in the future)

• Each position in the alignment has a structural conservation score, which represents the conservation in 3D space

• A position has a high conservation score if the aligned residues are spatially conserved


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Structurally Conserved Residues and Interface Residues

E.g. Residues with the top 20% of structure conservation scores (red) mapped to adrenodoxin (Adx, PDB code 1E6E:B) and known to bind adrenodoxin reductase (AR, blue).


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Training Dataset

• 274 non-redundant chains of heterocomplexes (<30% sequence identity) extracted from the PDB

• Each of these chains was accompanied with a structure alignment with at least 4 members


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SVM Training

A surface residue

↓

Sequence profile + ASA + Structural conservation score

in a window of 13 residues

(The residue to be predicted and 12 spatially nearest surface residues)

↓

Support vector machine classifier

↓

Interface or non-interface residue ?


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SVM Training

• Each residue was encoded as a feature vector with 13×21 dimensions: (the surface residue to be predicted + 12 nearest neighbors) x (20 amino acids + accessible surface area)

• Implemented using SVMlight with the radial basis function as a kernel. (γ = 0.01, regularization parameter C =10)

• A set of non-interface surface residues was randomly selected to make the ratio of positive and negative data 1:1

• 3 fold cross-validation was performed


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Predictor 1: Sequence profile + ASA.Predictor 2: Sequence profile + ASA + structural conservation scorePredictor 3: Sequence profile + ASA + raw structural conservation score without weighted by the normalized B-factor Predictor 4: Sequence profile + ASA+ normalized B-factor

The Performance of Various Predictors


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Precise prediction: at least 70% interface residues were identifiedPrecise prediction: at least 70% interface residues were identifiedCorrect prediction: at least 50 % interface residues were identifiedCorrect prediction: at least 50 % interface residues were identifiedPartial prediction: some but less than 50 % interface residues were Partial prediction: some but less than 50 % interface residues were identifiedidentifiedWrong prediction: no interface residues were identifiedWrong prediction: no interface residues were identified

The Performances of the Predictors


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Predicted Binding Sites - Example 1

Protein : domain 1 of the human coxsackie and adenovirus receptor (CAR D1)• Mediate adenoviruses and coxsackie virus B infection• CAR is an integral membrane protein expressed in a broad range of human and

murine cell type. CAR D1 is one of its two extracellular domains

Binding partner: knob domain of the adenoviruses serotype 12 (Ad12)


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Protein : adrendoxin (Adx) • In mitochondria of the adrenal cortex, the steroid hydroxylating system requires the

transfer of electrons from the membrane-attached flavoprotein AR via the soluble Adx to the membrane-integrated cytochrome P450 of the CYP 11 family

Binding partner: adrenodoxin reductase (AR)


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Protein : fibroblast growth factor receptor 2 (FGFR2) Ser252Trp Mutant

• Apert syndrome (AS) is caused by substitution of one of two adjacent residues, Ser252Trp or Pro253Arg

Binding partner: fibroblast growth factor (FGF2)


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Conclusions – Protein-protein Binding Sites

• Incorporating the structural conservation score improved the prediction performance of SVM significantly

• This study is an initial trial that exploits multiple structure alignment for the large scale prediction of functional regions

• We need better algorithms for multiple structure alignment (we have one benchmark for anyone interested)

• This method can be used to guide experiments, such as site-specific mutagenesis, or combined with docking procedures to limit the search space


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General Conclusions

• Using known features of protein structure these can be mapped to the corresponding sequences and used to train an SVM

• Having evaluated the SVM in a cross validation tests the performance can be determined

• Good performance is shown in training for both flexibility and sites of protein-protein interaction

• These predictors are currently being used to solve real biological problems

• Can this approach be applied to other aspects of structure?

1ytf

PUU: 1 Experts: 2

1d0gt

PUU: 1Experts: 3

1dgk

PUU: 6 Experts: 4

1aoga

PUU: 4 Experts: 3

1fohb

PUU: 2 Experts: 3

A. B.

C.D.

E.

Consider Domain Definitions:Holland et al. 2006 JMB Early Release

Veretnik et al. 2004 JMB 339(3), 647-678


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Challenge – Defining Domain Boundaries from Sequence

• A domain is the unit of currency of proteins – domain structures define function, indicate evolutionary relationships etc…

• Domain prediction from structure easier than from sequence, but still not a solved problem

• Recently developed an accurate test set of domain definitions and boundaries: http://pdomains.sdsc.edu

• Good luck!

Benchmark Data Available See:Holland et al 2006 JMB Early Release


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Acknowledgements

• Functional Flexibility– Jenny Gu & Michael

Gribskov

• Protein-protein Interactions– JoLan Chung & Wei Wang

• Domain Definitions– Stella Veretnik, Tim Holland,

Ilya Shindalov, Nick Alexandrov, Abdur Sikur

• Funding, NSF, NIH


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The structural conservation score

• Raw structural conservation score

where

if a is not gap and b is not gap otherwise

where N is the total number of aligned structures, si(x) is the amino acid at position x in the ith structure in the alignment, m is a modified PET substitution matrix calculated by Valdar et al.

N

i

N

ij

ji xsxsLNN

xC ))(),(()1(

2)(

))(),(()))(),((exp())(),(( xsxsMxsxsdxsxsL jijiji

)min()max(

)min(),(

0))(),(( mm

mbam

ji xsxsM


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The structure conservation score

• The B-factors determined by X-ray crystallographic experiments provide an indication of the degree of mobility and disorder of an atom in a protein structure

• Raw structural conservation scores were weighted by the normalized B-factors (Bnorm, i) to consider the structure flexibility

where

)()()( xrweighted xCxC

)exp()( , inormBxr

1 june 2015dimacs - machine learning in bioinformatics 1 machine learning as applied to structural...

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dimacs machine learning

protein function

predicting flexibility

proteinligand interaction

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structural bioinformatics

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spectrum of protein